Two-dimensional spectral imaging system

ABSTRACT

Systems, including methods, apparatus, and algorithms, for spectrally imaging a two-dimensional array of samples.

CROSS-REFERENCES

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 60/696,301, filed Jun. 30, 2005;and U.S. Provisional Patent Application Serial No. _(———————), filedJun. 29, 2006, titled “TWO-DIMENSIONAL SPECTRAL IMAGING SYSTEM,” andnaming Dar Bahatt and Chirag Patel as inventors. Each of these patentapplications is incorporated herein by reference in its entirety for allpurposes.

INTRODUCTION

The identity, amounts, properties, and/or interactions of samples, suchas samples including biomolecules (biological samples), can be analyzedby detecting light emitted by the samples. The emitted light can bedetected, and illumination optionally can be provided, by a suitablelight-detection system. To facilitate analysis of multiple samples withthe light-detection system, the samples can be disposed in an arraydefined by a sample holder. In some cases, more information about thesamples can be obtained by measuring distinct spectral components of thelight emitted by each sample in the array.

SUMMARY

The present teachings provide systems, including methods, apparatus, andalgorithms, for spectrally imaging a two-dimensional array of samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an exemplary spectral imaging system, inaccordance with aspects of the present teachings.

FIG. 2A is a graph of a pair of spectral profiles obtained fromtwo-dimensional spectral images of the same spectral composition imagedin an examination site of a spectral imaging system as a point source ordistributed across a well, in accordance with aspects of the presentteachings.

FIG. 2B is a schematic view of selected portions of another exemplaryspectral imaging system illustrating how an optical system of theimaging system resolves nondispersed and dispersed light from atwo-dimensional array of examination sites, in accordance with aspectsof the present teachings.

FIG. 3 is a schematic view of spectral imaging systems having variousexemplary arrangements of optical relay structures including short-passand/or long-pass filters, in accordance with aspects of the presentteachings.

FIG. 4 is a flowchart listing steps that can be performed with aspectral imaging system in an exemplary method of sample analysis thatincludes calibration, in accordance with aspects of the presentteachings.

FIG. 5 is a schematic view of selected portions of an exemplary spectralimaging system illustrating assignment of regions of interest on adetector using spectra from a luminophore(s) disposed in eachexamination site.

FIG. 6 is a schematic view of selected portions of another exemplaryspectral imaging system illustrating assignment of regions of interestusing filtered light, in accordance with aspects of the presentteachings.

FIG. 7 is a schematic view of selected portions of exemplary spectralimaging systems having diffraction gratings disposed at different anglesand having different line densities on each grating, in accordance withaspects of the present teachings.

FIG. 8 is a schematic view of an exemplary spectral imaging systemincluding a grism that positions spectra of interest generally on theoptical axis of the system, in accordance with aspects of the presentteachings.

FIG. 9 is a schematic view of an exemplary spectral imaging systemhaving a grating that positions spectra off the optical axis of thesystem, in accordance with aspects of the present teachings.

FIG. 10 is a side elevation view of selected portions of yet anotherexemplary spectral imaging system, particularly illustrating emittedlight rays traveling from spaced positions in an examination areathrough an optical system and to more closely spaced positions on adetector after optical reduction, in accordance with aspects of thepresent teachings.

FIG. 11 is a side elevation view of selected portions of a modifiedversion of the spectral imaging system of FIG. 10, in which theexamination area is disposed off-axis from the optical system anddetector, in accordance with aspects of the present teachings.

FIG. 12 is a side elevation view of selected portions of yet anothermodified version of the spectral imaging system of FIG. 10 that includesan exemplary illumination system, in accordance with aspects of thepresent teachings.

FIG. 13 is a partially schematic view of selected portions of anexemplary spectral imaging system that includes a pair of theillumination systems of FIG. 12, in accordance with aspects of thepresent teachings.

FIG. 14 is a plan view of the spectral imaging system of FIG. 13.

FIG. 15 a view of an exemplary spectral imaging system including anexemplary sample holder shown in plan and having a staggered arrangementof wells that results in a corresponding staggered array of spectralimages on a detector of the system, in accordance with aspects of thepresent teachings.

FIG. 16 is a sectional view of the sample holder of FIG. 15, takengenerally along line 16-16 of FIG. 15.

FIG. 17 is a sectional view of another exemplary sample holder, takengenerally as in FIG. 16, in accordance with aspects of the presentteachings.

FIG. 18 is an exploded view of an exemplary examination assembly forpositioning samples in an examination area of an exemplary spectralimaging system, viewed from generally below the assembly, in accordancewith aspects of the present teachings.

FIG. 19 is an exploded view of selected portions of the examinationassembly of FIG. 18, viewed from generally above the assembly.

FIG. 20 is a representation of exemplary image data collected by aspectral imaging system and of exemplary processing of the image data toidentify features of the image data, in accordance with aspects of thepresent teachings.

FIG. 21 is another representation of exemplary image data collected by aspectral imaging system and of additional or alternative exemplaryprocessing of the image data to identify features of the image data, inaccordance with aspects of the present teachings.

FIG. 22 is a representation of the image data of FIG. 21 illustratingexemplary conversion of the image data to a binary form by thresholding,to facilitate identification of feature boundaries within the imagedata, in accordance with aspects of the present teachings.

FIG. 23 is a schematic view of selected portions of a spectral imagingsystem that uses a fiducial to correct for variability of sampleholder/well position in an examination area of the system (and/orvariability of region of interest position on the detector), inaccordance with aspects of the present teachings.

FIG. 24 is a flowchart listing steps for performing an exemplary methodof sample analysis using a plurality of dyes, in accordance with aspectsof the present teachings.

FIG. 25 is a graph of an exemplary calibration matrix for a set of dyespectra detected in the same region of interest of a detector of aspectral imaging system, in accordance with aspects of the presentteachings.

FIG. 26 is a graph of the calibration matrix of FIG. 25 and exemplarysample spectra detected in the same region of interest, either near thebeginning or the end of thermal cycling for nucleic acid amplificationof a sample, in accordance with aspects of the present teachings.

FIG. 27 is a graph of the relative spectral contributions of the fourindicated dyes to the composite spectra of the sample of FIG. 26 at theindicated cycle numbers during thermal cycling of the sample to promoteamplification of nucleic acid template, if any, in the sample, inaccordance with aspects of the present teachings.

DESCRIPTION OF VARIOUS EMBODIMENTS

The present teachings provide systems, including methods, apparatus, andalgorithms, for spectrally imaging a two-dimensional array of samples.

The imaging systems described herein can include (1) an examination areaincluding a two-dimensional array of examination sites, (2) anillumination system, (3) an optical system (with at least anoutput/emission optical relay structure), and/or (4) a light detectorfor imaging, among others. The illumination system can include one ormore light sources that illuminate samples disposed in the examinationsites, to induce light emission by the samples. The optical system (oran output optical relay structure thereof) can include at least onedispersion element, such as a grating, that disperses the emitted light,particularly visible light, from each sample into a correspondingspectrum. In addition, the optical system can direct the dispersed lightonto a detection area of the detector such that a two-dimensional arrayof sample spectra (e.g., first order (+1 or −1) diffraction spectra)from the samples falls concurrently onto the detection area. In someexamples, the array of spectra, collectively, can be at leastsubstantially resolved from nondispersed (i.e., zero order) light fromthe samples, and, optionally, from higher order light, such as seconddiffraction order light from the samples. Accordingly, the opticalsystem can include one or more filters, such as low-pass and/orhigh-pass filters that filter the emitted light, to truncate eachspectrum at one or both ends. Selective truncation of spectra can enablethe optical system to expand a selected wavelength region of eachspectrum on the detector, for better resolution, while reducingcrosstalk between adjacent spectra that would be present withoutfilters. An overview of exemplary spectral imaging systems is presentedbelow in Section I.

The systems also can include a sample holder configured to be positionedin the examination area. The sample holder can have wells that holdfluid. The wells can be open (uncovered) or the sample holder caninclude one or more sealing elements that seal each well after placementof fluid (e.g., sample) in the well. The sample holders can define setsof orthogonal axes along which the wells are disposed. In addition, thewells can be disposed in an array of aligned rows and columns or canhave an offset relationship such that the wells are staggered. Theoptical system can define a dispersion axis that has any suitablerelationship to the array of spectra. In some embodiments, the array ofspectra can define a pair of orthogonal axes and the dispersion axis canbe parallel to one of the orthogonal axes or oblique to both orthogonalaxes. In some examples, an oblique spectral axis can offer a greaterdistance for extending spectra, to provide better resolution of spectralcomponents and/or less optical crosstalk. Further aspects of sampleholders and defining oblique spectral axes are described below, such asin Section III and Example 1, among others.

The methods can include calibrating a spectral imaging system to assignan array of regions of a detector corresponding to an array ofexamination sites. Calibration can include detecting light of knownspectral composition from the examination sites. The light of knownspectral composition can be provided from the examination sites, e.g.,from isolated/pure/unconjugated dyes, illumination light, test samples,and/or the like. The methods also or alternatively can include obtainingand/or processing spectral data from samples based on the assignedregions of interest. In some examples, the assigned regions of interestcan be moved based on detected signals from one or more fiducialsprovided by a sample holder. The fiducials can be one or more wells(e.g., to provide nondispersed light) and/or can be an opticallydetectable feature of the sample holder that is spaced from wells of thesample holder. Further aspects of calibrating spectral imaging systemsand analyzing samples with calibrated systems are described below inSections VIII and X, among others.

The methods also can include using a spectral imaging system to performkinetic assays of two-dimensional sample arrays. Each sample spectrumcan be monitored over time to determine a change, if any, in thespectrum. For example, each sample can include a plurality of reactionsthat are assayed and/or monitored over time by the spectral imagingsystem. In some examples, the reactions can include amplification ofnucleic acids to allow detection and/or quantitation of one or morenucleic acids in each sample. Further aspects of assays that can beperformed with spectral imaging systems are described elsewhere in thepresent teachings, such as in Section IX and in Example 9, among others.

The algorithms can be applied to image data collected by a spectralimaging system. In some examples, the algorithms can be used tocalibrate a detector and/or to identify spectral data for individualsamples. In some examples, the algorithms can, for example, reduceoptical distortion in spectral image data. In some examples, thedistortion can include stretching and/or curving of image data, amongothers. Alternatively, or in addition, the algorithms can selectparticular regions of spectral image data for processing such to providesoftware-based filtering of sample signals. Further aspects ofalgorithms that can be suitable for using in spectral imaging systemsare described, for example, in Examples 7, 8, 11, and 12, and in thepatent applications listed above under Cross-References, which areincorporated herein by reference.

Concurrent imaging of spectra from a two-dimensional array of samplescan have a number of advantages. For example, concurrent imaging cansave time and/or reduce mechanical complexities (such as vibrations,since it can obviate the need for changing spectral filters to determineemission wavelengths). Furthermore, concurrent imaging, in someexamples, can allow analysis of faster kinetic assays, since data can becollected during a kinetic assay from different samples at the same time(or in rapid succession) without movement of mechanical parts, providingfaster data acquisition and/or a better direct comparison between (orwithin) samples.

The following sections further describe these and other aspects of thepresent teachings, including (I) an overview of exemplary spectralimaging systems, (II) illumination systems, (III) examination areas andsample holders, (IV) optical systems, (V) detectors, (VI) controllers,(VII) thermal control systems, (VIII) calibration of spectral imagingsystems and sample analysis, (IX) applications, and (X) examples, amongothers.

I. Overview of Exemplary Spectral Imaging Systems

FIG. 1 shows a schematic view of an exemplary two-dimensional spectralimaging system 110. System 110 can include an illumination system 112,an examination area 114, an optical system 116 (e.g., with respectiveinput and output optical relay structures forming source optics 118 andemission optics 120) operatively coupled to the examination area, alight detector for imaging (an array detector) 122, a thermal controlsystem 124, and a controller 126, among others. These and other devices,assemblies, and systems described in the present teachings can be usedin any suitable combination and can be present any suitable number oftimes in a spectral imaging system.

The illumination system can include a light source 128 and source optics118. The light source can produce light for excitation of samplesdisposed in the examination area, and the source optics can direct thelight to the samples. In some examples, some or all of the opticalelements of the illumination system can be integral to the light source.The illumination system can illuminate examination area 114, which caninclude a two-dimensional array of examination sites 130. Standards orsamples 132 can be disposed in the examination area via a sample holder134 having a plurality of compartments, such as wells 136, forpositioning the standards or samples in the examination sites.

Light can be emitted from the examination sites, such as fromluminophores (e.g., fluorophores) disposed in the wells. The emittedlight can be directed to detector 122 by emission optics 120 forming a(linear or bent) optical path 138 between the examination sites and thedetector. The emission optics can include at least one dispersionelement 140 to spectrally disperse the emitted light. The emissionoptics also can include one or more filters 142 that selectivelyrestrict passage of part of the emitted light according to wavelength.Furthermore, the emission optics can direct the dispersed light onto adetection area 144 of the detector such that a two-dimensional array 146of spectra (also termed spectral images) of the two-dimensional array ofexamination sites falls concurrently on the detection area (if the sitesare illuminated at the same time).

Each spectrum can be a spectrally dispersed image of emitted light froma corresponding examination site, such as an image of spectral lightemitted from a sample disposed in an examination site. For example, inthe present illustration, each spectral image of the contents of acircular well results from dispersion of light in a direction parallelto a spectral axis 148 defined by the emission optics of the system, tostretch each well image into a generally oval shape (with a sufficientspectral range of emitted light). The intensity within each spectrum canbe determined by its spectral composition. For example, exemplaryspectrum 150 includes a pair of intensity peaks 152, 154 at distinctpixel positions (and thus at different wavelengths or wavelength ranges)along a line parallel to the spectral axis. Graph 156 illustrates asimplified one-dimensional spectral profile 158 that can be produced byprocessing image data for spectrum 150, by collapsing thetwo-dimensional spectral data from spectrum 150 into a linearvector/profile. In particular, the image data for spectrum 150 can besummed in a direction parallel to a spatial axis 160 orthogonal to thespectral axis.

Each sample spectrum can be detected by a corresponding region ofinterest 162 of the detector (and/or obtained as a portion of image datafrom the detector). The region of interest also can be abbreviated as aregion or “ROI” in the present teachings and is shown here as a dashedrectangle. Each region of interest can be assigned by calibration usingdata analysis instructions 164 (e.g., algorithms) of the controllerprior to analysis of test samples (e.g., in conjunction with calibrationstandards disposed in the examination sites) and/or can be assigned orrepositioned based on spectral images of the test samples themselves.The detector regions of interest can be elongate, with the length ofeach region disposed parallel to spectral axis 148 and the width of eachspectrum region perpendicular to the spectral axis. The axes of thearray of regions of interest can be aligned with orthogonal axes of thedetector, as shown here, or the array can be angularly offset (i.e.,slanted) on the detector. In some examples, image data from a slantedarray of regions of interest can be processed to effectively rotate theimage data relative to the matrix axes in which the image data isarranged. Such rotation of image data can facilitate further processingof the image data (such as collapsing two-dimensional image data in adirection(s) parallel to one or both orthogonal axes). Alternatively, orin addition, each region of interest can extend in a direction parallelto a spectral axis disposed obliquely to orthogonal axes defined by thedetector and/or the array of spectral images (e.g., see Example 1).Further aspects of processing slanted image data are described in U.S.Provisional Patent Application Ser. No. 60/696,301, filed Jun. 30, 2005,which is incorporated herein by reference.

The size and shape of each region of interest for a particular systemcan be defined by the light source, sample configuration, luminophore(s)in the samples, and/or the optical system, among others. In someexamples, these parameters can be adjusted such that the regions ofinterest for most or all of the samples do not overlap substantiallywith one another and also do not overlap substantially with adjacenthigher order or lower order diffraction (or nondispersed (zero order))regions. These parameters also can be adjusted such that the regions ofinterest (and thus the spectral images) are closely packed, which canprovide more efficient use of the detector (e.g., faster dataacquisition, higher signal-to-noise, etc.). For example, the width ofeach region of interest can be at least about the same as, or about two,five, or ten times greater than the lateral spacing (the gap) betweenthe adjacent sides of laterally adjacent regions of interest.Alternatively, or in addition, the length of each spectrum region can beat least about the same as, greater than, and/or at least about two,five, or ten times greater than the longitudinal spacing (the gap)between the adjacent ends of longitudinally adjacent regions ofinterest. Further aspects of calibrating spectral imaging systems,collecting image data, processing the image data, interpreting theprocessed image data, and determining assay results from the processedimage data are described elsewhere in the present teachings, such as inSections VIII and X, among others, and in the patent applications listedabove under Cross-References, which are incorporated herein byreference.

Thermal control system 124 can be operatively coupled to the examinationarea for controlling the temperature thereof. In some embodiments, thethermal control system can be configured for thermal cycling of theexamination area and particularly a sample holder/wells disposedtherein. The thermal cycling can be performed in an automated manner forone cycle or a plurality of cycles. In addition, a thermally conductiveportion of the thermal control system can be configured to be in contactwith the sample holder, for conductive heating and/or cooling thereof.Further aspects of thermal control systems are described elsewhere inthe present teachings, such as in Section VII and in Example 6, amongothers.

The components of spectral imaging systems provided by the presentteachings can, more generally, be selected and/or configured accordingto application, desired level of automation, and so on. For example,controller(s) 126 can be configured to control any suitable componentsof the system, including illumination system 112, examination area 114,optical system 116, detector 122, thermal control system 124, and/or thelike. In chemiluminescence (including bioluminescence) applications, theimaging system can include the components indicated above, and/or othercomponents, without the light source and/or source optics, or with thelight source and/or source optics present but not used.

The spectral imaging system also can include additional components, suchas (1) a sample-handling mechanism to convey samples and/or sampleholders to and/or from the examination area, (2) a registration deviceto drive movement of the sample holder within the examination area,and/or (3) a sample-identification mechanism such as a barcode reader ora radiofrequency (RF)-tag reader to identify samples and/or sampleholders, and optionally to configure or operate system componentsaccordingly, among others.

FIG. 2A shows a graph 165 of a pair of one-dimensional spectral profiles166, 167 obtained from two-dimensional spectral images of the samespectral composition imaged in an examination site of a spectral imagingsystem. Narrower spectral profile 166 can be produced from a sampleconfigured as a point (or line) source, such as spotted in a very smallarea (as on a biochip), restricted to a very small volume (such as in oron bead), imaged with an aperture of small size, and/or occupying asmall portion of the field of view, among others. Broader spectralprofile 167 can be produced from the same sample distributed across anarea or in a volume of significant size for the optical arrangement ofthe system, such that the sample does not act as a point (or line)source. In particular, the sample can be disposed in a sample holder ofthe imaging systems described herein. The sample holder can have wellslarge enough to substantially affect the shape of the resulting spectrumdetected. For example, spectral profile 167 can be broadenedsubstantially in directions parallel to the spectral axis relative tospectral profile 166 from the same sample arranged as a point source, asindicated by comparing the full width at half maximum (FWHM) for eachprofile, indicated at 169 and 170, respectively.

The spectral profile and two-dimensional spectral image detected from awell can be produced by convolving the well image size and/or shape withthe spectrum that would be produced from a point or line source in placeof the well. Accordingly, each point arranged along a line parallel tothe spectral axis in a spectral image of the well does not correspond toa single wavelength from the sample, but to a range of wavelengthsdetermined by the sample/well size and/or shape (and the system optics).In some examples, calibration of the spectral imaging system thus canmeasure, at least in part, how each well alters and/or broadens spectralimages and/or profiles measured from the well relative to a point/linesource.

FIG. 2B shows selected portions of another exemplary spectral imagingsystem 180. System 180 can have an optical system 182 with a diffractiongrating 184 that disperses light from a two-dimensional array ofexamination sites 186 to form a series of corresponding arrays ofdifferent diffraction orders, e.g., arrays 188-194 of spectra 196.(Additional arrays (e.g., −3, +3, 4, +4, etc.) can be formed anddetected but are not shown here to simplify the presentation.) Arrays188-194 can flank a zero order array 198 of nonspectral images producedby nondispersed light from the examination sites passing through thegrating without being diffracted.

The distance between the arrays can be adjusted according to the linespacing of the grating, as specified by the grating equation.Accordingly, the grating can be selected to provide an array of spectraof the same order in which the array is collectively resolved fromadjacent arrays, using light of a selected wavelength range. Forexample, the grating can be selected such that the array of +1 (or −1)diffraction order spectra is collectively resolved from nondispersed(zero order) light and/or from +2 (or −2) diffraction order spectra.Accordingly, a detector 200 of the imaging system can be positioned suchthat each spectrum 196 of one of the diffraction orders (+1 here) fallson the detector concurrently. In some examples, the detector can bepositioned also to detect one or more adjacent spectra, such as one ormore zero order images, indicated by a dashed extension of the detectorat 202, to serve as a fiducial(s) to correct for variability in samplepositioning and/or optical system alignment (e.g., see Example 8). Ahigher order array (such as +2 or −2) can be detected instead (or inaddition). However, the signal intensity generally decreases for higherorder arrays relative to lower order arrays.

FIG. 3 shows selected portions of a series of spectral imaging systemshaving various exemplary optical systems with distinct arrangements ofoptical relay structures, particularly spectral filters.

Imaging system 220 can include an optical system 222 with a grating 224that disperses light received from an array of examination sites 226.The light can, for example, be visible light of about 400 nm to 800 nmand/or light of a wavelength range determined by selection of a suitableluminophore(s) and/or light source. The optical system also can directthe dispersed light to a detector 228 as a corresponding array 230 ofspectra 232 disposed in an overlapping arrangement on the detector. Inparticular, adjacent ends of the spectra, indicated at 234, can overlap(generally the longer wavelength end of one spectrum overlapping theshorter wavelength end of an adjacent spectrum along a dispersion axis).An overlapping arrangement, as used herein, is any arrangement in whichthere is significant interference or crosstalk between adjacent spectrasuch that analysis of the spectra is impaired.

Imaging system 220 can be modified to form an imaging system 240 with anoptical system 242 including a short-pass filter 244. The short-passfilter can selectively restrict passage of light of longer wavelengths,to truncate each spectrum at one end, indicated at 246. The short-passfilter can be sufficient to effectively remove the crosstalk betweenadjacent spectra. Alternatively, sufficient overlap and crosstalk canstill be present, indicated at 248.

Imaging system 220 alternatively can be modified to form an imagingsystem 260 with an optical system 262 including a long-pass filter 264.The long-pass filter can selectively restrict passage of light ofshorter wavelengths, to truncate each spectrum at the other end,indicated at 266. The long-pass filter can be sufficient to effectivelyremove the crosstalk between adjacent spectra. Alternatively, sufficientoverlap and crosstalk can still be present, indicated at 268. The systemcan have only one long-pass filter or can include at least a firstlong-pass filter (such as a beam splitter) that selectively removesexcitation light from emitted light and a second long-pass filterfollowing the first long-pass filter in the optical path and configuredsubstantially exclusively to receive emitted light (relative toexcitation light) and to truncate the spectrum of the emitted light.

Imaging system 220 alternatively can be modified to form an imagingsystem 280 with an optical system 282 including both short-pass filter244 and long-pass filter 264. The filters in combination can truncateeach spectrum at opposing ends to substantially reduce crosstalk betweenthe spectra, indicated at 284.

The filters can be disposed at any position(s) in the optical system,preferably in a collimated beam zone of the optical path (such as afterthe objective lens or before the detector lens in the optical path). Forexample, each filter can be disposed before (upstream of) or after(downstream of) the dispersion element in the optical path for emittedlight. Furthermore, the filters can be separate components or can becombined in a single component as a relatively broad spectrum band-passfilter. The filters can select any suitable range of wavelengths. Inexemplary embodiments, the long-pass filter can selectively permitpassage of light having wavelengths of longer than about 450 nm to about550 nm, or of longer than about 500 nm; the short-pass filter canselectively permit passage of light having wavelengths of shorter thanabout 550 to about 700 nm, or of shorter than about 600 to about 650 nm;and both filters, if present together, can selectively permit passage ofabout a 50-200 nm or about a 100-150 nm range of wavelengths and/or canrestrict passage of light having wavelengths of less than about 500 nmand greater than about 650 nm.

The spectral imaging system can be of any suitable size and weight andcan be used in any suitable environment. For example, the system canhave a size and weight (e.g., greater than about 5,000 cc and/or greaterthan about 10 kg) that restricts ready transport of the system, suchthat the system can be relatively permanent once installed.Alternatively, the system can have a size and weight (e.g., less thanabout 5,000 cc and/or less than about 10 kg) that facilitatesportability, and/or a size and weight (e.g., less than about 1000 cubiccentimeters and/or less than about 2 kg) that permits the system to behand-held. Accordingly, the system can be used in medical applications,(e.g., for biodefense, forensics, paternity conflicts, clinicaldiagnosis, and/or the like) as a portable and/or hand-held medicaldevice that can be transported readily to various testing sites.

II. Illumination Systems

An illumination system, as used herein, generally comprises anymechanism for producing light capable of illuminating, and/or inducing asuitable or desired response from, a sample. For example, when used inan optical assay, light from the illumination system can, as a result ofilluminating a sample, produce emitted (e.g., photoluminescence) light(using an excitation light source), transmitted light, reflected light,and/or scattered light, among others. These different forms of light canbe present exclusively, or in various combinations, and can includeultraviolet, visible, and/or infrared light, among others.

The illumination system can have one or more light sources. Each lightsource can include continuous wave and pulsed lasers, arc (e.g., xenon)lamps, incandescent (e.g., tungsten halogen) lamps, fluorescent lamps,electroluminescent devices, laser diodes, and/or light-emitting diodes(LEDs), among others. Such light sources can be capable of use in one ormore illumination modes, including continuous and/or time-varying (e.g.,pulsed or sinusoidally varying) modes, among others, depending on systemconfiguration and/or intended application. For example, an arc lamp orcontinuous wave laser can be used to provide continuous illumination,and a pulsed laser or pulsed LED can be used to provide intermittentillumination. Such light sources also can produce coherent, incoherent,monochromatic, polychromatic, polarized, and/or unpolarized light, amongothers. For example, an arc lamp can be used to provide (at leastinitially) incoherent, polychromatic, unpolarized light, and a laser canbe used to provide (at least initially) coherent, monochromatic,polarized light, among other possibilities.

The light source can be selected, at least in part, based on thespectrum of light produced. Generally, the light source can producelight (e.g., ultraviolet and/or visible light) capable of exciting thesamples (and particularly luminophores therein) for light emission.Accordingly, the light source(s) can be selected according to itsability to produce light of a wavelength, wavelength range, and/or setof wavelength ranges that is absorbed by and excites one or moreluminophores in the samples. In some examples, the light source(s) canbe selected for its ability to excite every luminophore of a set or twoor more luminophores disposed in the samples, such that each luminophoredetectably emits light.

The light source(s) can be disposed in any suitable position relative tothe sample holder and emission optics. For example, the light source canbe disposed on the same side of the sample holder as the emission opticsor can oppose the emission optics across the sample holder. If disposedon the same side as the emission optics, the light source can bedisposed substantially on-axis (i.e., near or on an optical axis definedby the emission optics) or can be disposed substantially off-axis. Anon-axis light source can be achieved, for example, by disposing aplurality of light sources around the optical axis, oriented to directexcitation light toward the sample holder. An off-axis light source canbe achieved, for example, via an optical element(s), that bends abeam(s) from the light source to bring the beam coincident with theoptical axis.

The illumination system can include a light source(s) used incombination with various source optics (such as any combination of theoptical elements described below in Section IV). The source optics canbe used to alter the nature of the light output by the light source(e.g., its color (spectrum or chromaticity, such as via filtering),intensity, polarization, and/or coherence, among others). Alternatively,or in addition, the source optics can be used to direct and/or alter thesize, shape, and/or numerosity of the light beam(s) (e.g., to illuminateselected locations in an examination area with one or more light beams).Furthermore, the source optics can include a lighthomogenizer(s)/mixer(s) to increase the uniformity of illumination ofthe examination area. Alternatively, or in addition, the source opticscan include a collection optical element(s) to increase the percentageof light from the light source that is directed to the examination area.In some examples, the collection optical element can be a light guidethat operates by total internal reflection. The light that is ultimatelyincident on the sample(s) can be produced by one or more light sources,and can be directed and/or modified, optionally, by one or more opticalelements operatively disposed between the light source(s) and theexamination area. The resultant light beam or beams can be one or moreof various forms, including but not limited to diverging, collimated,and converging, among others.

The illumination system can be configured to illuminate an array ofsamples in an examination area concurrently or at different times. Ifilluminated at different times, the samples can be serially illuminatedone by one, line by line (e.g., one row or one column at a time), and/orsection by section (e.g., two or more rows or columns at once), and/orthe like. Furthermore, if illuminated at different times, subsets of asample array can be illuminated with distinct light sources (e.g.,operated serially), and/or the same light source directed serially ontothe subsets, among others.

In some examples, the illumination system can have at least two lightsources (and/or illumination assemblies) that illuminate samples withdistinct wavelengths of light. For example, the light sources caninclude a light source that produces light of shorter wavelengths, suchas a blue LED, and a light source that produces light of longerwavelengths, such as green LED. The at least two light sources orassemblies can be selected, for example, to excite different dyes in thesamples, based on different absorption spectra of the different dyes.The light sources or assemblies can be turned on at the same time or canbe turned on serially. In some embodiments, excitation light of thelonger-wavelength source can be removed from the emission optical pathvia a notch filter in the emission optics.

Further aspects of illumination systems that can be suitable forspectral imaging systems of the present teachings are describedelsewhere in the present teachings, such as Example 5, and in the patentapplications listed above under Cross-References, which are incorporatedherein by reference.

III. Examination Areas and Sample Holders

The imaging systems of the present teachings can include an examinationarea operatively disposed in relation to the optical system. Inparticular, the examination area can be positioned such that theexamination area is illuminated effectively by the illumination systemand imaged effectively by the detector for emitted light dispersed bythe emission optics. The examination area (defining an object plane) canbe defined adjacent or on a receiver structure, such as a platform orstage. The receiver structure can be configured to receive (andoptionally retain) a sample holder and position the sample holder suchthat samples of the sample holder are disposed in the examination area,particularly in a two-dimensional array of examination sites of theexamination area. The examination sites can be defined substantially bythe positions in which the samples are disposed in the examination area.Alternatively, or in addition, the examination sites can be predefinedin correspondence with regions of interest of a detector of an imagingsystem, such as by calibration.

A sample holder (a sample-support device), as used herein, generallycomprises any mechanism for supporting one or more samples inexamination sites of an examination area. In typical embodiments, thesample holder allows for the receipt and/or transmission of lightrelative to one or more samples supported by the device. Generalexamples of suitable sample holders can include trays, wells, tubes,containers, channels, chambers, frames, carriages, holders, slides,shelves, stages, housings, and/or the like. Specific examples ofsuitable sample holders can include microplates, PCR plates or cards,cell culture plates, biochips, hybridization chambers, chromatographyplates or columns, and/or microscope slides, among others.

The sample holders can be configured to allow top detection (e.g., byhaving an open or at least partially transparent top), bottom detection(e.g., by having an at least partially transparent bottom), and/or sidedetection (e.g., by having an at least partially transparent side),among others.

Each sample holder can provide a two-dimensional array of locations orcompartments for supporting samples. The array can have any suitablearrangement of the locations/compartments, including rectilinear (with aplurality of rows and columns), circular, irregular, and/or the like.(In some examples, a ring-light illuminator can be suitable forillumination of a circular arrangement of wells.) Specific locations inthe sample holder, such as wells in microplates, PCR plates/cards, andcell culture plates, and array sites on biochips, can comprise assaysites. For example, microplates (and/or PCR plates/cards, microtiterplates, and/or cell culture plates) can include arrays of 6, 12, 24, 48,96, 384, 864, 1536, 3456, and/or 9600 such assay sites, among others.

The sample holders and/or arrays suitable for spectral imaging systemscan have standard or nonstandard features. Assay sites (e.g., wells) canbe arrayed with a uniform spacing or a nonuniform spacing. For example,all rows and all columns of the wells can be aligned or a subset of therows and/or columns can be offset from the others (such as in astaggered arrangement). In some embodiments, the spacing betweenadjacent rows relative to the spacing between adjacent columns can bedifferent. For example, the spacing between assay sites along a sampleholder axis corresponding to a spectral axis of the system can begreater than their spacing orthogonal to this sample holder axis. Thisarrangement can provide more efficient use of the detector. For example,excess lateral spacing (corresponding, generally, to wasted detectorarea) between laterally adjacent spectral images can be minimized whileproviding a sufficient longitudinal dimension for dispersion of spectralcomponents of each spectral image.

The assay sites of a sample holder can have any suitable shape and size.The sites can be two-dimensional, such as areas on a surface (generallydisposed in fluid communication), or can be three-dimensional, such aswells (generally disposed in fluid isolation). In any case, each sitecan have any suitable shape in a plane defined by the sample holder,such as circular, polygonal (e.g., rectangular), elliptical, oval,and/or the like. In some examples, each site can be elongate in theplane of the sample holder. Elongate sites can provide a more line-likeemission of light from the sites. The elongate sites thus can beoriented with the long axis of each site orthogonal to a sample holderaxis corresponding to a spectral axis of dispersion. This orientationcan provide more line-like spectral components and thus betterresolution (less overlap and/or greater spacing) between spectralcomponents within each spectral image. If three-dimensional, each sitecan be a well having any suitable three-dimensional shape, such ascylindrical, semi-spherical, conical, frustoconical, and/or polyhedral,among others. Furthermore, each well can have any suitable depthrelative to the well's area. Relatively deeper wells can be suitable,for example, to increase the volume of sample that can be held by eachwell and/or to reduce the angle of light emission from samples disposedin the wells (e.g., with samples occupying only a lower portion of thewells). Relatively shallower wells can be suitable, for example, toincrease the angle of light emission from samples disposed in the wells.Overall, the wells can provide compartments of any suitable size andvolume. For example the wells can be about 0.1 to 10 mm or about 0.5 to5 mm in diameter and/or can have a volume of at least about 0.1 μL,about 0.1 to 10 μL, or about 0.5 to 5 μL, among others.

The sample holder can be covered and/or sealed hermetically with asealing element to restrict evaporation of fluid from samples disposedin the assay sites, particularly if the samples are heated. For example,the sample holder can be covered/sealed with a cover for the entirearray, and/or individual assay sites (or sets of assay sites) can becovered/sealed individually or in sets with caps or plugs.

Further aspects of sample holders are described elsewhere in the presentteachings, such as in Example 6, and in the patent applications listedabove under Cross-References, which are incorporated herein byreference.

IV. Optical Systems

Optical systems, including input (illumination) and/or output (emission)relay structures, as described herein, generally comprise anymechanism(s) for directing, transmitting, and/or conducting light withinthe imaging systems such as from a light source toward a sample (orexamination area) and/or from a sample (or examination area) toward adetector. The optical system can include optical relay structures thatstand alone and/or that are integral to other system components, such asthe light source and/or detector, among others. Each optical relaystructure can be configured to direct one or more light beams, in thesame or different directions, along the same, multiple, or differentoptical paths.

A. Overview of Optical Relay Structures

Each optical relay structure can include any suitable combination of oneor more optical elements. These elements independently can be part of asingle (e.g., excitation or emission) relay structure, or can be sharedbetween two or more relay structures. Exemplary optical elements caninclude (1) reflective elements, such as concave, planar, and/or convexmirrors, among others, (2) refractive elements, such as converging,diverging, concave, convex, and/or planoconvex lenses, includingcircular and/or cylindrical lenses, among others, (3) transmissive orconductive elements, such as glass or quartz fiber optics and/or liquidlight guides, (4) diffractive elements such as gratings or otherdispersion elements, and/or (5) subtractive elements (such as filters),among others.

The optical relay structure(s) can be selected, in conjunction with thelight source(s) and/or detector(s), to allow any suitable or desiredcombinations of illumination and/or detection. For example, thesecomponents can be arranged to allow same-side, (locally) anti-parallelor straight-on (“epi”) illumination and detection, such as topillumination and top detection, or bottom illumination and bottomdetection, respectively. Alternatively, or in addition, these componentscan be arranged to allow opposite side, (locally) parallel orstraight-through (“trans”) illumination and detection, such as topillumination and bottom detection, or bottom illumination and topdetection, respectively. Alternatively, or in addition, these componentscan be arranged to allow illumination and/or detection at obliqueangles. For example, illumination light can impinge on the bottom of asample holder at an acute angle (e.g., about 45 degrees) relative todetection. Such oblique illumination and detection can reduce the amountof excitation light reaching the detector, relative to straight-on episystems (light source and detector directed at about 90 degrees tosample holder) or straight-through trans systems (light source directedthrough a sample holder directly at a detector). Epi systems areespecially suitable for photoluminescence assays, trans systems areespecially suitable for absorbance assays, and oblique systems (with theincidence angle set above the critical angle) are especially suitablefor total internal reflection assays, among others.

B. Dispersion Elements

An optical dispersion element (a color separator), as used herein,generally comprises any mechanism for dispersing light spatiallyaccording to its wavelength composition.

The dispersion element can use any suitable mechanism(s) and/orcomponent(s) for dispersing light. Exemplary mechanisms can includediffraction, interference, and/or refraction, among others. Exemplarycomponents can include (diffraction) gratings, interferometers, and/orprisms, among others. In some embodiments, the dispersion element caninclude two or more sequentially acting components, employing the sameor different mechanisms, with the first component achieving a coarsecolor separation, and the second component achieving a finer or finalcolor separation.

Gratings can have any suitable structure. The gratings can betransmission gratings and/or reflection gratings, among others.Furthermore, the gratings can have a planar or nonplanar surface atwhich light is received. Exemplary nonplanar surfaces can includeparabolic, toroidal, and/or spherical surfaces, among others. Thegratings can have any suitable spacing of lines (generally, grooves toprovide ruled gratings) and/or fringes (holographic gratings), accordingto the application. A suitable spacing of lines and/or fringes can bedetermined theoretically (e.g., by using a diffraction grating equationand/or empirically for a particular system configuration). Generally,the spacing can be decreased (e.g., the number of lines/mm increased) toincrease the amount of spectral dispersion (e.g., to lengthen eachspectral image on the detector and/or to increase the spacing betweenadjacent spectra of the same or different diffraction orders) or thespacing can be increased for the opposite effect. In some examples, thegrating can be a blazed grating, that is, a grating optimized for aparticular wavelength (or spectral region) and/or a particular angle ofincidence (or range of angular incidences).

The dispersion element can separate multi-wavelength light by directinglight with different wavelengths along different paths (e.g., indifferent directions, at different angles, etc.) The separation can bepartial or complete, and can create bands or beamlets of light, whichcan be continuous or discrete, and which can be partially overlapping orcompletely distinct. The character of the separation typically will bedetermined at least in part by the character of the light beingseparated. Thus, input light with several well-spaced wavelengthcomponents can give rise to separated output light at several discrete(well-spaced) positions, while input light with closely or continuouslyspaced wavelength components can give rise to separated output lightover a continuous set of positions. The character of the separation alsocan be determined at least in part by the shape of the sample (e.g., asdetermined at least in part by the volume and/or area of the sample andthe shape of the well supporting the sample). For example, a samplehaving a shape that deviates from a point or line source can tend toproduce a corresponding deviation from a point or line in the shape ofthe dispersed spectral components (e.g., excitation of a sample in acircular well can produce circular (or oval) spectral components in aspectral image of the sample). More generally, any deviation from apoint or line source can produce a spectrum in which the entire spectrumand spectral components thereof are broadened to reduce the resolutionbetween adjacent spectra and/or between spectral components within thespectrum.

An imaging system with a particular well size/shape can provide anysuitable degree of broadening of a corresponding spectrum relative to aspectrum of the same spectral composition produced by a point source inplace of a well. The broadening can be by a distance on the detectorcorresponding to a spectral distance of at least about 10 nm or 20 nm,among others, as measured for the width of a peak within the spectrum athalf maximum (FHWM) or at 10% of maximum intensity. For example, anexemplary peak having a width measured at half maximum intensity (FWHM)of 30 nm can be broadened to at least about 40 nm or 50 nm. Accordingly,absolute wavelengths can be difficult to measure with the spectralimaging system because light of a single wavelength from the sample maynot fall as a sharp line or point on the detector but can be spread outin opposing directions parallel to the spectral axis according to thesample size and shape.

The separated light generally can form any distinguishable pattern.Thus, the separated light can form a linear array, in which the averagewavelength of light varies with position along the linear array.

The separated light can be directed onto a common detector, ontoseparate detectors, or onto a combination of detectors. The relationshipbetween wavelength and position on the detector(s) can be determinedempirically, for example, using input or calibration light of knownwavelength(s). Alternatively, or in addition, the relationship betweenwavelength and position on the detector(s) can be determinedtheoretically, for example, by calculating the optical paths for lightof different wavelengths. The position(s) of light on the detector canbe determined by a variety of factors, including (1) the mechanism usedto separate the light, (2) the angles at which the light enters andleaves the dispersion element, (3) the wavelength(s) of the light, (4)the distance between the dispersion element and the detector(s)(generally, greater distances between the dispersion element anddetector(s) will give rise to greater separations between light ofdifferent wavelengths on the detector(s)), (5) the magnification (oroptical reduction/demagnification) of the system, (6) the focal lengthof the detector lens, and/or (7) the type of pattern (e.g., linearversus circular) formed by the separated light, among others.

The spatial distribution or pattern of detected light can be convertedinto information about the amount (including presence/absence),distribution, identity(ies), structure, and/or the like of components ofthe sample, using any suitable method. These methods can include simplylooking up a result in a look-up table (e.g., position (x,y) on thedetector corresponds to light of wavelength λ (or wavelengths withinsome extended range (e.g., λ₁ to λ₂)) emitted from position (X,Y) (orpositions within some extended range) in the sample holder, evaluating afunction expressing the relationship between these parameters, and/orthe like. The desired result can be obtainable simply by notingqualitatively the presence or absence of light at a particular positionon the detector (subject, in some cases, to some threshold amount), orit can be obtainable by determining quantitatively the amount of light(intensity, number of photons, amount of energy, etc.) detected at theposition, among others.

The dispersion element can be disposed, in some embodiments, so that itacts only on light directed from a sample toward a detector, withoutacting on (or, in most cases, even contacting) light directed from alight source toward a sample.

C. Miscellaneous Optical Elements

The spectral imaging system, and/or components thereof, also can includemiscellaneous optical elements capable of performing additional and/orduplicative optical functions. These optical elements can include (1)intensity filters (such as neutral density filters) for reducing theintensity of light, (2) spectral filters (such as interference filters,diffraction gratings, a colored material such as colored glass, and/orprisms) for altering or selecting the wavelength(s) of light (e.g., forseparating longer-wavelength emission light from shorter-wavelengthexcitation light, in single-photon photoluminescence, for defining themaximum wavelength of emitted light that reaches the detector, and/orfor separating shorter-wavelength emission light from longer-wavelengthexcitation light, in multi-photon photoluminescence), (3) polarizationfilters (such as “polarizers”) for altering or selecting thepolarization of light, (4) “confocal optics elements” (such as anaperture or slit positioned in an intermediate image plane) for reducingor eliminating out-of-focus light, (5) beam collimators for convertinginput light (particularly diverging input light) into an at leastsubstantially collimated light beam, (6) beam expanders for increasingthe cross-sectional area of a beam of light, (7) beam homogenizers (suchas a fiber optic cable or liquid light guide) for enhancing the spatialuniformity of light, and/or (8) reference monitors for correcting forvariations (e.g., fluctuations and/or inhomogeneities) in light producedby a light source and/or other optical elements. These elements can befunctional in one or more of the space, time, and/or frequency domains,as necessary or desired.

A spectral filter element generally acts to reduce or eliminate emissionat selected wavelengths and/or over selected ranges of wavelengths(i.e., to reduce or remove undesired spectral components). Thesefunctions can be accomplished via any suitable mechanism, using a singlesubtractive element or a combination of subtractive elements. Suitablesubtractive elements include filter elements such as (1) short-pass(cut-off) filters, which selectively pass short-wavelength light andreject long-wavelength light, (2) long-pass (cut-on) filters, whichselectively pass long-wavelength light and reject short-wavelengthlight, (3) band-pass filters, which selectively pass light with aparticular wavelength (or range of wavelengths) and reject light withlower and higher wavelengths, and/or (4) band-reject (or notch) filters,which reject light with a particular wavelength (or range ofwavelengths) and pass light with shorter and longer wavelengths, amongothers. Short-pass and long-pass filters (also know as edge filters) canbe characterized by a cut-on or cut-off wavelength, among others, andband-pass and band-reject filters can be characterized by a centerwavelength and a bandwidth, among others. Suitable subtractive elementscan include thin-film (e.g., metallic and/or interference) coatings,colored filter glass, holographic filters, liquid-crystal tunablefilters, and/or acousto-optical tunable filters, among others. Thesesubtractive elements can work by absorbing, reflecting, and/or bending(refracting or diffracting) light, among others. In some embodiments,the subtractive element can work by filtering portions or all of theexcitation light, either before or after the excitation lightilluminates samples. Filtering portions before sample illumination canbe performed, for example, on portions which, when absorbed, give riseto undesired spectral components of the emission.

The relative positions of any intensity, spectral, polarization, and/orother optical elements generally can be varied without affecting theoperation of the spectral imaging system. In addition, if there is morethan one optical path, for example, to permit top and bottom or obliqueillumination and/or detection, optical elements can be shared and/orused independently in each path. The particular order, positions, andcombinations of optical elements for a particular experiment can dependon the apparatus, the assay mode, and the sample (target material),among other factors. In some cases, optical elements can be associatedwith an exchange mechanism, such as a wheel or slider, that allowsconvenient and automatable placement and exchange of optical elements byrotating, sliding, or otherwise bringing preselected optical elementsinto or out of the optical path. In some examples, optical elements canbe associated with an adjustment mechanism (automated or manual), forexample, to adjust the axial position and/or alignment of opticalelements, and/or to increase or decrease magnification (or opticalreduction) of images, among others.

V. Detectors

A detector, as used herein, generally comprises any mechanism fordetecting light transmitted or otherwise originating from a sample andoptionally converting the detected light into a representative signal.

Exemplary detectors can include film, charge-coupled devices (CCDs),complementary metal oxide semiconductor (CMOS) devices, intensifiedcharge-coupled devices (ICCDs), charge injection device (CID) arrays,vidicon tubes, photomultiplier tubes (PMTs), photomultiplier tube (PMT)arrays, position sensitive photomultiplier tubes, photodiodes (such asphotodiode arrays), and/or avalanche photodiodes, among others. Suchdetectors can be capable of use in one or more detection modes,including (1) imaging and point-reading modes, (2) discrete (e.g.,photon-counting) and analog (e.g., current-integration) modes, and/or(3) steady-state and time-resolved modes, among others. The detectorscan be configured to receive a two-dimensional array of light, which canbe separated parallel to a first dimension according to position in asample or sample array, and parallel to a second dimension according toposition and spectral composition. Toward this end, the detector caninclude bins (e.g., regions of interest and/or sub-regions with theregions of interest) for detecting light from different samples and/orof different colors from the samples, for example, correspondingsubstantially to light from different luminophores. These bins can bethe same or different sizes, and can be formed of one or a plurality ofsub-bins, or pixels (sensor elements), depending in part on the averageseparation between spectral peaks outputted by the color separator.

The detector can be used alone or in combination with various opticsand/or other mechanisms (such as the optical relay structures describedabove). These optics and/or other mechanisms can be used to alterproperties of the light (e.g., color, intensity, polarization,coherence, and/or size, shape, and/or numerosity of the light beam(s),as described elsewhere herein), prior to its detection. In someembodiments, the detector can be part of or coupled to a spectrograph orspectroscope for analyzing the spectral composition of the detectedlight.

VI. Controllers

A controller, as used herein, generally comprises any mechanism forcontrolling components and/or other aspects of a spectral imagingsystem. These components and/or other aspects can include the lightsource(s), optical relay structures, registration device, fluidicsmechanism, thermal control device, detector, and/or data analysis, amongothers. For example, the controller can determine and/or change (1) thewavelength, intensity, and/or (spatial and/or temporal) uniformity,and/or spatial position/direction of light produced by the light source;(2) the order and timing of sample delivery by the registration deviceand image acquisition by the detector; (3) the wavelength and/orintensity of light detected by the detector; (4) the temperature ofsamples disposed in or near the examination area; (5) the relativetiming of temperature regulation, light source actuation, and detectorexposure/detector actuation; and/or (6) the composition of samples in ornear the examination area, among others. The controller can includehardware, software, firmware, and/or a combination thereof, and can beany device, or combination of devices, adapted to store and executeinstructions to control associated imaging system components. Thecontroller can include one or more of various devices, such as acomputer, computer server, microprocessor, memory, logic unit, and/orprocessor-based system capable of performing a sequence of logic and/orarithmetic operations. In addition, processing can be centralized (withtwo or more components sharing a common controller) and/or distributed(with one or more components having their own dedicated controllers,acting alone, or connected to one another and/or a central controller).

The controller also or alternatively can be configured to processsample/image information detected by the detector. For example, thecontroller can (1) identify image regions corresponding to individualsamples and/or individual sample spectral components, and/or (2) relatethe information detected by various regions/pixels/bins of the detectorto particular samples, spectral components, and/or assays, among others.

VII. Thermal Control Systems

A thermal control system, as used herein, generally comprises any systemfor regulating the temperature of samples. The thermal control systemcan be configured to add heat to samples (e.g., including a heater(s))and/or to remove heat from samples (e.g., including a cooler(s)). Thethermal control system also can include a temperature sensor configuredto measure temperature adjacent (or in) the samples. The thermal controlsystem further can include or be coupled to a controller that uses themeasured temperature to determine when, how long, where, and/or at whatlevel, among others, to actuate the heater and/or cooler, to achieve andmaintain a suitable target temperature or suitable profile of targettemperatures for the samples.

The heater (or heaters) and/or cooler (or coolers) can operate by anysuitable mechanism and can have any suitable structure. Exemplarymechanisms for heating and/or cooling can be by conduction (i.e., bycontact), convection (i.e., through air), and/or radiation (i.e., viawaves and/or particles). Exemplary heaters/coolers can include resistiveelements, Peltier devices (e.g., solid state devices that operate by athermoelectric effect using a thermocouple(s)), infrared lamps,refrigeration units (e.g., by gas expansion/compression), fluidcirculators (e.g., fans or fluid conduits), and/or the like.

The temperature sensor (or sensors) can have any suitable structure andcan be a contact or noncontact device. Exemplary temperature sensors caninclude thermocouples, thermistors, resistance temperature devices,radiation thermometers (pyrometers), thermal imagers, (liquid in glass)thermometers, and/or the like.

The thermal control system can be disposed in any suitable positionrelative to the sample holder/samples. For example, the thermal controldevice can be disposed below, above, laterally (i.e., adjacent one ormore sides), and/or substantially enclosing the sample holder (e.g., toprovide a heating/cooling chamber). In exemplary embodiments, thethermal control system is disposed below the sample holder and includesa conductive member that contacts the bottom of the sample holder toprovide conductive heating/cooling.

The thermal control system can be controlled to provide any suitabletemperature profile for the samples, and the profile can be the same ordifferent for each sample of a sample holder. For example, the thermalcontrol system can be configured to rapidly heat or cool samples.Exemplary rates of rapid heating and/or cooling include a rate oftemperature change of at least about 10° C. per minute, or at leastabout 1, 2, 5, or 10° C. per second. In some embodiments, the thermalcontrol system can maintain the temperature of the samples at a constanttemperature (e.g., below, around, and/or above room temperature). Insome embodiments, the thermal control device can change the temperatureof the samples during an analysis (e.g., to test the effect of differenttemperature on the samples and/or to promote chemical reactions (e.g.,enzyme-catalyzed reactions) and/or binding reactions (such as binding orunbinding) in the samples, among others).

VIII. Calibration of Spectral Imaging Systems and Sample Analysis

The spectral imaging systems of the present teachings can be calibratedand used for analysis of test samples at any suitable relative times andin any suitable fashion. For example, the imaging systems can becalibrated before, during, and/or after sample analysis, as describedfurther below.

A spectral imaging system can be calibrated by any suitable approach atany suitable time(s) during and/or after the manufacture of the system.Accordingly, calibration can be performed before an imaging system issold to a customer and/or can be performed at the site(s) of operationby a user (including service personnel). On-site calibration can beperformed to calibrate the system for the first time or as are-calibration of the system to adjust for changes in the systemconfiguration. On-site calibration can be particularly suitable for aportable system, which can be subject to more frequent mechanicalshocks, which can alter the alignment of system components.

FIG. 4 shows an exemplary flowchart 340 listing steps that can beperformed in a method of sample analysis that includes calibration(e.g., the first two steps listed in the flowchart). The steps can beperformed in any suitable order, in any suitable combination, and anysuitable number of times. Furthermore, the steps can be combined in anysuitable way with other steps described elsewhere in the presentteachings to perform other methods of sample analysis.

Dispersed light of known spectral composition can be directed from oneor more sites of a two-dimensional array of examination sites onto adetection area, shown at 342. Known spectral composition, as usedherein, means that the light has at least one spectral feature of knownwavelength (such as an intensity peak or valley, among others), or thatat least substantially every spectral feature of the light is known. Thelight can be emitted from a standard source and/or a test sample to beassayed. The standard source or test sample can include one or moreluminophores (e.g., fluorescent dyes (fluorophores)). Alternatively, thelight can be excitation light (e.g., from a light source(s) of theillumination system) that is filtered or unfiltered. The light of knownspectral composition can be directed concurrently to the detection area(e.g., for calibration with only one dye, only a dye mixture, or onlyone configuration of excitation light). In other examples, differentspectral components or combinations of spectral components can bedirected to the detection area at different times (e.g., to performcalibration using two or more dyes placed serially into the examinationarea of the imaging system).

A corresponding array of regions of interest can be assigned for thedetection area, shown at 344. The array of regions can be assignedrelative to the detection area based on detection of dispersed light ofknown spectral composition from any suitable number of examinationsites, including only one, at least a pair, a two-dimensionalarrangement, at least most, or all of the examination sites.Accordingly, each region of interest can be assigned based on detectionof light of known spectral composition from a corresponding examinationsite. Alternatively, a subset of the regions of interest can be assignedwithout detecting light of known spectral composition from correspondingexamination sites. For example, if dyes are used for calibration, thedyes may not be placed in every well of a sample holder disposed in theexamination area (and/or can be in wells that are ignored forcalibration to simplify data processing). In this case, the array ofregions can be generated, at least in part, by interpolation,extrapolation, and/or by positioning a predefined array of regions inrelation to the detection area based on signals detected from one ormore sentinel examination sites.

Spectral data can be obtained for samples disposed in the examinationsites based on the regions of interest, shown at 346. The samples can bethe same test samples used for calibration or can be test samplesdisposed in examination sites after calibration standards are removedfrom the examination sites. Using test samples for calibration can besuitable if the test samples provide a known spectral composition at theoutset of a reaction, such as in a kinetic reaction that monitorschanges in spectral data from samples over time. The spectral data canbe obtained in exact correspondence with the assigned regions ofinterest, for example, selecting a portion of the detected data definedby a region of interest and/or selectively detecting spectral data foreach sample using a corresponding region of interest of the detector.Alternatively, the positions the regions of the interest can be adjustedbefore the spectral data is obtained for individual samples. Forexample, the regions of interest can be moved translationally and/orpivotally based on detection of a fiducial(s) (see Example 8) and thenappropriate portions of the detected data can be selected and/or the new(adjusted) regions of interest used to selectively detect spectral datafor individual samples.

The spectral data can be processed for each sample, shown at 348.Processing can include simplifying the spectral data, such as summingthe spectral data orthogonal to the dispersion axis, to create aone-dimensional vector from two-dimensional data. Alternatively, or inaddition, processing can include binning portions of the spectral datafor a sample. A part or all of the spectral data for a sample (and/orfor a region of interest) can be placed into uniform bins (such as binsthat are two, three, four, five, etc.) pixels wide (in a dimensionparallel to the spectral axis) and processed as part of the bin (e.g.,summed). Alternatively, or in addition, a part of all of the spectraldata for a sample (and/or for a region of interest) can be placed intononuniform bins, such as bins centered around intensity peaks in thespectral data, and processed as part of the bin (e.g., summed). Forexample, each expected intensity maxima in the spectral data for asample or detector region can have its own bin(s). In addition,processing can include removing background. Processing also can includedetermining the amount (including presence/absence) of a luminophoresignal for each sample. Moreover, processing can include correlating theamount determined with a sample aspect, to provide information about thesample. Furthermore, processing can include detecting a change, if any,in spectral data for a sample over time, to provide kinetic data for areaction or interaction occurring (or not occurring) in the sample. Thechange can be related to time and/or to cycle number in repetitivethermal cycling, among others.

FIG. 5 shows selected portions of an exemplary spectral imaging system360 illustrating assignment of regions of interest 362 on a detector364. One or more luminophores (“dyes”) 366 can be disposed in eachexamination site 368. In some examples, the same set of one or moreluminophores can be disposed in one or more, most, or all of theexamination sites, such as by disposing the luminophores via a sampleholder having wells in which the luminophores are contained. In someexamples, the same set of luminophores is disposed in most or all of theexamination sites, optionally in substantially the same relative amountsand/or in known amounts. If a set of luminophores is disposed in theexamination sites, the luminophores can be disposed individually (e.g.,serially) and/or as one or more mixtures of two or more or all of theluminophores of the set. The luminophores can be the same luminophoresto be used for assays with distinct test samples or can include at leastone or more (or be all) luminophores that are distinct from those usedin test samples.

Each region of interest 362 can be assigned based on a spectrum 370 (orspectra) produced by the luminophore. For example, here, spectrum 370can allow assignment of an associated region of interest 362 accordingto a detected intensity maximum 372, minimum, threshold, transition,and/or the like. Furthermore, the region of interest can be assigned atleast in part based on a predefined size of the region of interest. Thepredefined size can be based, at least in part, on a predefined ormeasured pixel to wavelength relationship on the detector along aspectral axis. For example, in exemplary embodiments, light can bedispersed at about 1 nm of wavelength per pixel on the detector.Accordingly, an approximate wavelength range 374 (ignoring spectralbroadening due to the sample size/shape) can be selected based on thepixel length of the region of interest. For example, the region ofinterest can have a predefined length of about 50-200 pixels and apredefined width of about 10-50 pixels, among others, which can bepositioned on the detector based on the detected signal from aluminophore(s) disposed in a corresponding examination site (add/orbased on detection of a light beam from the site; see below).Alternatively, the length and/or width of the region of interest can beset based on the detected signal from the luminophore (and/or based ondetection of a light beam from the site; see below).

FIG. 6 shows selected portions of another exemplary spectral imagingsystem 380 illustrating assignment of regions of interest 382 usingbeams of light from examination sites (wells) 384. The beams can becreated by a light source 386 and, optionally, at least one filter 388disposed between the light source and the examination sites and/orbetween the examination sites and the detector. The light source can bepart of the illumination system used to excite samples for lightemission or can be a distinct light source used for calibration, such asa light source of narrow wavelength without a filter or a light sourceof broader wavelength with a filter. In some examples, the filter can bea band-pass filter that permits passage of a relatively narrowwavelength range of light. For example, here the filter produces anarrow enough wavelength range that light from each examination site isdispersed to produce a substantially circular well image 390 on thedetector, with only slight elongation. Each region of interest 382 canbe assigned according to the position of spectral well image 390 and/oraccording to a plurality of spectral well images created with differentband-pass filters (e.g., a pair of band-pass filters to position eachend of the region of interest). In some examples, the filter can permitpassage of a range of wavelengths generally corresponding to opposingend positions of the region of interest (see FIG. 3).

IX. Applications

Spectral imaging systems in accordance with the present teachings can beused for any suitable purposes, such as detecting and/or monitoring theoccurrence of, and/or changes in, light received from one or moresuitable samples.

The detection or monitoring of light can be performed qualitativelyand/or quantitatively. Qualitative detection can include measurement ofthe presence or absence of a signal, and/or a change in a signal frompresent to absent, or absent to present, among others. Here, presence orabsence can be in reference to a whole signal (such as any light) and/ora component of the signal (such as light of a particular wavelength (orwavelength region), polarization, and/or the like). Quantitativedetection can include measurement of the magnitude of a signal, such asan intensity, wavelength, polarization, and/or lifetime, among others.The quantified signal can be used alone and/or compared or combined withother quantified signals and/or calibration standards. The standard cantake the form of a calibration curve, a calculation of an expectedresponse, and/or a control sample measured before, during, and/or aftermeasurement of a test sample.

The detected or monitored light can be used for any suitable purpose,for example, to determine the amount, concentration, activity, and/orphysical properties (including interactions) of an analyte (such as aphotoactive analyte) in a sample. Here, the analyte can be the actualsubstance of interest and/or a reporter substance that reports on theactual substance of interest.

The substance of interest can be a reaction component. Exemplaryreaction components can include an enzyme, enzyme substrate, enzymeproduct, and/or enzyme modulator (e.g., agonist and/or antagonist).Suitable reactions can occur in vivo and/or in vitro, for example, aspart of a cell-lysis experiment and/or a polymerase chain reaction (PCR)preparation. Exemplary reaction components also can include precursorsand/or products of a synthetic pathway, such as an amino acid, peptide,protein, nucleotide, polynucleotide, carbohydrate, fatty acid, lipid,and/or the like.

The substance of interest also can be the subject and/or product of aseparatory process, such as on a chromatograph, gel, column, and/or thelike. Here, the separatory process can include single processes, such ascolumns giving rise to fractions, and/or multiple processes, such asparallel lanes on a gel giving rise to sets of bands.

The substance of interest also can be the subject of a sequencingprocess, such as a peptide, protein, and/or nucleic acid (e.g., RNAand/or DNA) sequencing process. Here, the sequence can include aminoacid sequence, nucleotide or base sequence (e.g., G, C, T, A, U, etc.),and so on, and the sequencing process can include generating fragments(or other derivatives) of the substance to be sequenced and labelingthose fragments (before or after their generation) with differentluminophores. Thus, in nucleic acid sequencing, the presence of a G, C,T, A, or U at a particular position in a substance of interest, or in afragment or derivative thereof, can be determined by the identity of anassociated luminophore.

The substance of interest also can be the subject of an identification,or affinity, process, such as a northern, western, and/or southern blot.

In some cases, the effect of some condition on the substance of interestcan be determined, for example, by comparing results in the presence ofthe condition with predicted and/or measured results in the absence ofthe condition and/or the presence of another condition. Exemplaryconditions can include presence or absence of a modulator (agonist orantagonist) or cofactor, and/or changes in temperature, concentration,pH, osmolarity, ionic strength, and/or the like.

The sample can include any appropriate material, with any suitableorigin. For example, the sample can include a biomolecule, organelle,virus, cell, tissue, organ, and/or organism. A sample optionally can bea biological sample, such as a sample including test material fromblood, urine, saliva, and/or mucous, among others. A sample optionallycan be an environmental sample, such as a sample including test materialfrom air, water, or soil, among others. A sample can be aqueous, and cancontain biologically compatible organic solvents, buffering agents,inorganic salts, or other components known in the art for assaysolutions. Suitable samples (or compositions) can include compounds,mixtures, surfaces, solutions, emulsions, suspensions, cell cultures,fermentation cultures, cells, suspended cells, adherent cells, tissues,secretions, and/or derivatives and/or extracts thereof.

The spectral imaging systems can be employed to assay samples, andparticularly reactions within the samples, kinetically (i.e., at one ormore times before the end of each reaction) and/or at steady state(i.e., after the endpoint of each reaction). With kinetic assays, thesystems can monitor reactions in real time during the course of thereactions. In some examples, the spectral imaging systems can monitortwo or more reactions within each sample concurrently to perform amultiplexed analysis. In particular, the progress of each reactionwithin a sample can be measured/monitored by a change(s) in lightemission from the sample in a characteristic spectral region. Forexample, changes in emission from a distinct luminophore correspondingto each distinct reaction can be measured. The change in emission duringthe reaction can be caused by any suitable mechanism, including creationof the luminophore, degradation of the luminophore, structuralmodification of the luminophore, a change in the environment around theluminophore (such as its spacing from a donor or quencher), and/or achange (increase/decrease) in the energy transfer efficiency of anenergy transfer pair including the luminophore.

In exemplary embodiments, the spectral imaging systems can be employedto detect and/or quantify two or more different nucleic acids (e.g., DNAor RNA) in each sample. For example, the nucleic acids can be quantifiedaccording to the rate at which they can be amplified detectably from thesample by an amplification reaction in which the nucleic acids arecopied exponentially and/or linearly. Any suitable amplificationapproach can be used, including approaches that rely on thermal cycling(such as the polymerase chain reaction (PCR)) and/or that aresubstantially isothermal (such as Nucleic Acid Sequence-BasedAmplification (NASBA), Loop-Mediated Isothermal Amplification (LAMP),Rolling Circle Amplification (RCA), Self Sustained Sequence Replication(S3R), Strand Displacement Amplification (SDA)), and/or the like. Insome cases, probes/primers for different nucleic acid analytes in asample can be labeled with a different luminophore and a quencher/energytransfer partner of the luminophore. Hybridization of a probe/primer tothe analyte (e.g., with a molecular beacon probe) and/or cleavage (suchas enzymatically) of the luminophore and/or quencher as a result ofhybridization can produce a change in light emission from theluminophore. A suitable assay, the TaqMan® assay, that can quantifynucleic acids according to the rate of change in light emission isavailable from Applied Biosystems. Further aspects of amplificationbased assays are described below in Example 9.

The apparatus and methods described herein generally can be used withany suitable optically active composition (sample), for any suitablepurpose. These compositions can include light-emitting,light-transmitting, light-absorbing, light-scattering, and/orlight-reflecting compositions. Exemplary compositions can include singlemoieties and/or mixtures of two or more moieties capable of producinglight at one or more wavelengths. The ability to separate colorsdescribed herein is particularly useful for single moieties that emit attwo or more distinguishable wavelengths, and/or mixtures of moietiesthat collectively emit at two or more distinguishable wavelengths.

Exemplary compositions can be photoluminescent and/or chemiluminescent,among others. Photoluminescent sources produce photoluminescence lightin response to illumination with suitable excitation light.Photoluminescence can include fluorescence (i.e., light produced by asinglet-to-singlet electronic transition) and/or phosphorescence (i.e.,light produced by a triplet-to-single electronic transition), amongothers. Chemiluminescent sources produce chemiluminescence lightassociated with a chemical reaction (e.g., as part of a reaction thatproduces an intermediary or product in an excited electronic state thatsubsequently decays by production of light). Chemiluminescence caninclude bioluminescence (i.e., light produced by a biological reaction),among others.

Exemplary compositions can be naturally and/or artificially occurring.Naturally occurring compositions can include green fluorescent protein(GFP), phycobiliproteins, luciferase, and/or their many variations,among others. Artificially occurring compositions can include, forexample rhodamine, fluorescein, FAM™/SYBR® Green I, VIC®/JOE,NED™/TAMRA™/Cy3™, ROX™/Texas Red®, Cy5™ and/or semiconductornanocrystals, among others. Suitable natural and artificial compositionsare disclosed in the following publication, among others, which isincorporated herein by reference: Richard P. Haugland, Handbook ofFluorescent Probes and Research Chemicals (6^(th) ed. 1996).

X. EXAMPLES

The following examples describe selected aspects of the presentteachings, including exemplary systems, methods, and algorithms forspectral imaging of samples. These examples are included forillustration and are not intended to limit or define the scope of thepresent teachings.

Example 1 Exemplary Dispersion Element Configurations

This example describes exemplary spectral imaging systems 420, 450, 480having distinct configurations of dispersion elements; see FIG. 7.

Imaging system 420 can include a dispersion element, namely a grating422, that disperses light from a two-dimensional array of wells 424 toproduce a corresponding array 426 of spectra 428 detected by a detector430. The spectra can be arranged in rows and columns parallel toorthogonal axes 432, 434 defined by the array of spectra and, if thearray is not slanted, aligned with corresponding detector axes. Grating422 can have lines 436 oriented to produce a spectral axis 438 that isparallel to one of the axes (here, axis 432) of array 426. Accordingly,the long axes of spectra 428 can be aligned along a row or column ofspectra, limiting the length of a spectrum according to the spacing ofspectra measured parallel to one of the orthogonal axes (here, measuredbetween corresponding spectral positions along a row).

Imaging system 450 can include a grating 452 that is rotated relative tograting 422 of system 420, to define a spectral axis 454 that is obliqueto orthogonal axes 456, 458 defined by an array 460 of spectra 462produced from the wells. Accordingly, grating 452 can have a higherdensity of lines 464 than grating 422 such that spectra 462 extendfarther parallel to the spectral axis than spectra 428 of system 420extend parallel to spectral axis 438. Furthermore, spectra 462 can bealigned diagonally within the array to limit the length to which eachspectrum can extend without overlap.

Imaging system 480 can include a grating 482 that is rotated relative togratings 422 and 452 of respective systems 420 and 450, to define aspectral axis 484 that is oblique to the orthogonal axes defined by thearray of spectra 486. Accordingly, grating 482 can be have a higherdensity of lines 488 than grating 452 such that the spectra extend evenfarther than spectra 462, for example, between spectra in an adjacentrow, as shown here.

Example 2 Exemplary Spectral Imaging System with a Grism

This example describes an exemplary spectral imaging system 500including a grism 502; see FIG. 8.

System 500 can disperse light emitted from an array of wells 504 anddirect the dispersed light to a detector 506 to form a correspondingarray of spectra 508 on the detector. The system can include anobjective lens (or lens assembly) 510, filters 512, 514, grism 502, anddetector lens (or lens assembly) 516 (e.g., a CCD camera).

The objective lens can receive emitted light from the wells, generallydiverging light, and direct the light to long-pass filter 512 andshort-pass filter 514, to select a suitable range of emitted light fordispersion. The objective lens can substantially collimate the lightsuch that the filters receive collimated light. In some embodiments, thefilters can be disposed elsewhere in the optical path, such asdownstream or flanking the grism.

Grism 502 can include a grating 518 and one or more prisms, such asflanking symmetrical prisms 520, 522. The grating can disperse thefiltered light and the prisms can shift the filtered/dispersed lightlaterally (and/or change the light angle), such that the dispersed lightof interest remains substantially on optical axis 524 of the system.

Detector lens 516 can focus the dispersed, laterally shifted light ontodetector 506. Nondispersed light can be disposed off (lateral to) theoptical axis and can be received by a lateral region of the detector orcan be undetected.

Example 3 Exemplary Spectral Imaging System with Off-Axis Imaging

This example describes an exemplary spectral imaging system 540 thatdetects spectra off-axis; see FIG. 9.

Imaging system 540 can include an optical system 542 generallyconfigured as described above for imaging system 500 (see FIG. 8).However, instead of a grism, the optical system can include only agrating 544. Accordingly, dispersed spectra 546 imaged by a detector 548of the system can be disposed lateral to the optical axis 550 of thesystem. In contrast, nondispersed (zero order) light can be imagedsubstantially on optical axis 550, if suitable.

Example 4 Exemplary Spectral Imaging Systems with Optical Reduction

This example describes exemplary spectral imaging systems that opticallyreduce dispersed sample images; see FIGS. 10 and 11.

FIG. 10 shows selected portions of an exemplary spectral imaging system570 with an on-axis configuration designed generally as indicated inExample 2. System 570 can include an examination area 572, a detector574, and an optical system 576 operatively disposed relative to theexamination area and the detector. In particular, the optical system canreceive light from the examination area, collimate the light, filter thelight, spectrally disperse the light, and re-focus the light onto thedetector to produce optically reduced spectral images of the samples. Inexemplary embodiments, the spectral images can be reduced about 2- to10-fold or about 5-fold relative to the samples being imaged.

Examination area 572 can include a sample holder 578 including samples,such as samples 580, 582 disposed in an array. Emitted light from eachsample, indicated at 584, 586 can be resolved by the optical system suchthat spectral images of the samples are correspondingly resolved on thedetector, indicated at 588, after optical reduction.

Optical system 576 can include an objective lens assembly 590, afilter-grism assembly 592, and a detector lens assembly 594. Eachassembly can employ a plurality of optical elements (or alternativelycan be only a single element).

FIG. 11 shows selected portions of an exemplary spectral imaging system610 that is a modified version of system 570 (see FIG. 10). System 610can include a reflective optical element 612 that permits examinationarea 614 to be disposed off-axis from optical system 576 and detector574.

Example 5 Exemplary Spectral Imaging Systems with Illumination Systems

This example describes exemplary spectral imaging systems that includeillumination systems; see FIGS. 12-14.

FIG. 12 shows a spectral imaging system 630 that is a modified versionof systems 570 and 610 (see FIGS. 10 and 11). System 630 can include anillumination system 632 that illuminates examination area 634. Theillumination beam can travel through a dichroic mirror 636 to a sampleholder 638 disposed in the examination area, back to dichroic mirror, tooptical system 576, and finally detector 574. The dichroic mirror can beconfigured to selectively reflect emitted light to optical system 576(toward the detector) and to selectively transmit excitation light thatis reflected back from the sample holder, to separate excitation andemission light.

Illumination system 632 can include a light source 640, such as alight-emitting diode 642, and source optics 644 that collect,homogenize, and focus the excitation light from the light-emitting diode(such that the cross-sectional size and/or shape of the light beam ischanged in size and/or shape to substantially match the size and shapeof the examination area). In exemplary embodiments, the source opticscan include a collection element(s), a homogenization element, and anysuitable additional optical elements such as refractive lenses 654, 656and a filter 658, among others. In exemplary embodiments, the lightsource can be a LUXEON® III Blue LED.

FIGS. 13 and 14 show selected portions of an exemplary spectral imagingsystem 680 that is a modified version of system 630, with a plurality ofillumination assemblies 682, 684 each corresponding to illuminationsystem 632 of FIG. 12. Each illumination assembly can include a lightsource, such as light-emitting diode 642 and source optics 644. However,excitation light 686 from each light source (rays from only the frontlight source are shown here to simplify the presentation) can bedirected to a sample holder 688 via respective mirrors 690, 692, whichcan be symmetrically offset angularly from dichroic mirror 636 (and/or aspectrally nonselective mirror)(see FIG. 12 also). Emitted light 694from the sample holder can be reflected by dichroic mirror 636 toemission optics 576 and ultimately to detector 574 (see FIG. 10 also).Each light assembly 682, 684 can illuminate the sample holder from anysuitable angle 696 relative to an orthogonal axis 698 from an imageplane defined by the sample holder (see FIG. 14). In some examples,angle 696 can be about 20-45 degrees, with exemplary embodiments havingangles of 25, 30, 35, or 40 degrees (and thus a total angle betweenlight excitation beams of 50, 60, 70, or 80 degrees). The imaging systemthus can have two or more light assemblies of the same structure butdisposed at different positions.

Example 6 Exemplary Sample Holders and Examination Assemblies

This example describes exemplary sample holders and examinationassemblies that can be suitable for holding samples in an examinationarea of the spectral imaging systems of the present teachings; see FIGS.15-19.

FIG. 15 shows an exemplary spectral imaging system 710 including anexemplary sample holder 712 having a frame 714 with a staggeredarrangement of wells 716 disposed in the frame. System 710 can include adispersive optical system 718 that directs an array 720 of spectralimages 722 of the wells to a detector 724. Exemplary spectral components725 in one of the spectral images are shown here to improve clarity. Thestaggered arrangement can offer more distance between adjacent spectralimages as measured parallel to a spectral axis 726 of the imagingsystem, than a corresponding nonstaggered arrangement of the wells.Accordingly, each spectral image can be lengthened by greaterdispersion, without substantial overlap of adjacent (end-to-end)spectral images, for better resolution of spectral components withineach spectral image.

The wells of the sample holder can be arranged in lines (e.g., columnsin the present illustration) disposed parallel to orthogonal axes 728,730. The orthogonal axes can be parallel (or oblique) to the perimeterof the frame. Wells in parallel lines can be offset from one another byany suitable fraction of the spacing between wells within the lines. Forexample, here, the wells in every other line (column) 732, 734 areoffset from wells in the remaining lines (columns) 736, 738 by one-halfthe spacing between adjacent wells within the line.

The wells can have any suitable spacing measured parallel to each ofaxes 728, 730. For example, the wells can have a greater spacingmeasured parallel to one of the axes (here, axis 730) than measuredparallel to the other axis (here, axis 728). For example, the wells canhave about two, three, four, etc. times the spacing measured parallel toone of the axes relative to the other axis. This arrangement can allowassignment of more elongate rectangles as regions of interest on thedetector. Furthermore, the spacing between adjacent lines of wells canbe about the same as (or different than) the spacing of wells withineach line. In some examples, the spacing between adjacent lines of wellsand within each line can be a standard defined by the Society forBiomolecular Screening (SBS) as a suitable center-to-center spacing formicroplate wells. Accordingly, the spacing can be 9 mm, 4.5 mm, or 2.25mm, among others. This spacing can offer substantial advantages forfluid dispensing into the wells using automated (or manual) dispensersconstructed according to an SBS standard. In exemplary embodiments, theline-to-line spacing (from center to center) is 4.5 mm, the diameter ofeach well is 2.0 mm with a volume capacity of about 1.5 μL, and thesample holder has a total of 24 wells in four lines/columns.

FIG. 16 shows a sectional view of sample holder 712. The sample holdercan include a body or body layer 750 defining an array of cavities 752corresponding to wells 716. A cover layer 754 can be disposed over thecavities to provide a sealing element 756 for each well. Fluid access tothe well can be provided by a channel 758 formed between the cover layerand the body. The channel can be in communication with a dedicated portfor inputting fluid contents 760 into the well or can be incommunication with a network of channels that extends to some or all ofthe other wells of the sample holder, for input of fluid contents to aplurality of wells via a common inlet port. In any case, the channel canbe closed, and the well sealed, by a pin or stake 762 that engages thecover layer and deforms the cover layer, shown in phantom outline andindicated at 764, such that the cover layer contacts an apposed surfaceof the body. Stake 762 can extend upward (rather than downward as shownhere) such that the sample holder is inverted relative to theconfiguration of FIG. 16 before placement in an examination area (e.g.,see FIGS. 18 and 19).

In some embodiments, reagents can be dispensed into the wells before thecover layer is assembled with the body. For example, fluid dispensingequipment can be used to dispense aliquots of the reagents into thewells from above the body. In some examples, the reagents can then bedried. In any case, the cover layer can be assembled with the body, toprotect the contents of the wells. The cover layer can be any suitablematerial, such as a pressure-sensitive adhesive layer.

FIG. 17 shows a sectional view of another exemplary sample holder 780.The sample holder can include a body 782 defining an array of cavitiesor wells 784. Each well can be sealed by the same sealing element 786,which can be placed over the body after a sample 788 is disposed in eachwell. The sealing element can have a plug region 790 that fits into thewell or can have a flat inner surface, among others. In otherembodiments, the wells each can have a corresponding sealing elementformed as a separate component.

FIG. 18 shows an exemplary examination assembly 810 for positioningsamples in an examination area of an exemplary spectral imaging system,viewed from generally below the assembly, with the assembly in anexploded configuration. The examination assembly can include a receiver812, a sample holder 814, a mask 816, and a window assembly 818disposed, respectively, in a vertical arrangement as shown here. Thewindow assembly can be optically transmissive to excited and/or emittedlight, to permit epi-illumination of the sample holder through thewindow assembly. In addition, the window assembly can urge the receiver,the sample holder, and mask together, such as by applying a weight tothe mask, to maintain consistent engagement between these structures,for optical and/or thermal purposes, among others.

FIG. 19 shows receiver 812, sample holder 814, and mask 816 in anexploded configuration, as viewed from above and to the side of thesestructures.

Receiver 812 can provide a stage on which the sample holder ispositioned. Accordingly, the receiver can define an examination area 820in which the sample holder is received. The receiver thus can have agenerally planar top surface with projections extending upwardtherefrom. The projections can include positioning pins 822 received inrespective openings 824, 826 of the sample holder and mask, to restrictlateral movement of the sample holder and mask. The projections also caninclude an array of stakes 827 (also see stake 762 of FIG. 16)positioned to block channels in the sample holder (e.g., a channelsimilar to channel 758 of FIG. 16) when the sample holder is engagedwith the receiver. The receiver can be a thermally conductive member,such as a metal plate or block. Accordingly, the receiver can beoperatively coupled to a thermal control system, to transfer thermalenergy between the thermal control system and the sample holder, forheating and cooling the sample holder, thereby providing temperatureregulation of the samples therein.

Sample holder 814 can provide an array of wells 840. The wells can beaccessed by a common inlet port 842 (see FIG. 18) for addition of thesame test material to some or all of the wells. Accordingly, the portcan communicate with some of all of the wells by a network of channels844 (e.g., similar to channel 758 of FIG. 16), which can be closed bystakes 827 when the sample holder is aligned with the stakes by matingwith pins 822 and urged against receiver 812. In some embodiments, thesample holder can have at least two inlet ports, such as a first inletport for introduction of a test material, and at least a second inletport for introduction of a control material. The sample holder can berectangular, with a thickness that is substantially less than its lengthand width, to provide a card-shaped holder.

Mask 816 can provide an array of light-transmissive apertures 846. Theapertures can be arranged in alignment with wells 840 of the sampleholder when the mask is mated with the receiver via pins 822. The maskcan be opaque around the apertures, to avoid detection of opticalsignals from positions between the wells of the sample holder (e.g.,from channels 844). The mask can be a relatively thin, opaque plate withan array of cylindrical through-holes. Alternatively, the mask can bethicker, as shown here, with apertures that are frustoconical orcylindrical through-holes, among others. Accordingly, with a thicker (orthinner) mask, the opaque regions can be formed using an opaque materialto form the body of the mask. Alternatively, the apertures can beoptical apertures, rather than through-holes, formed, for example, byapplying an opaque coating to one or both sides of a light-transmissivebody, without creating through-holes, to form a patterned array ofopenings in the coating.

Example 7 Exemplary Algorithms for Assigning Regions of Interest

This example describes exemplary algorithms for processing image data toassign regions of interest; see FIGS. 20-22.

FIG. 20 shows an image representation of image data 860 collected by anexemplary spectral imaging system. Image data 860 can describe an arrayof spectral images or spectra 862 (e.g., first order images) of wells ofa sample holder holding the same dye in an examination area of theimaging system.

One or more algorithms can be used to process the image data foridentification of features within the image data. The features can bemaxima, minima, transitions, and/or thresholds, among others, within theimage data and can be spectral (and/or nonspectral) features produced bydispersed (and/or nondispersed) light. The features can be arranged in afeature array in correspondence with a sample/well array that producedthe features. Accordingly, the algorithms can search for features basedon predefined parameters regarding expected relative feature positionswithin the image data, to reduce identification of spurious features.

The image data can be collapsed in a direction parallel to one or moreaxes to facilitate feature identification. For example, the image datacan be collapsed (summed) parallel to a first orthogonal axis 864, toprovide a profile 866 that facilitates identification of centerpositions 868 of rows of features within the image data, as intensitymaxima. Alternatively, or in addition, the image can be collapsed(summed) parallel to a second orthogonal axis 870, to provide a profile872 that facilitates identification of column positions 874 of spectralmaxima within the image data. Feature maxima 876 thus can be identifiedapproximately at the intersection of the row and column positionsdetermined.

Each profile 866, 872 can be processed in any suitable way to obtain themaxima. For example, the local maxima of these profiles can found byfirst smoothing the profile (e.g., by using a Savitzky-Golay filter, anaveraging filter, etc.) and applying a peak-finding algorithm (e.g.,finding where the slope is zero), to find the approximate coordinateswithin the image data for each spectrum. The approximate coordinates canbe refined by doing a local search for the area of highest intensityaround these row and column positions. Further aspects of searching forlocal maxima iteratively (e.g., to identify centroids and/or intensitymidpoints), are described in the patent applications listed above underCross-References, which are incorporated herein by reference.

Approximate and/or refined feature positions within the image data canbe used to assign regions of interest within the image data and thusrelative to a detection area that detected the image data. For example,features 876 can be used to assign the locations of one or both sidesand/or ends of regions of interest that include the features. Theboundaries of each region of interest can be assigned by, for example,setting the boundary at predefined numbers of pixels from the featureposition parallel to axes 864 and 870 (such as based on a calculated ormeasured wavelength per pixel relationship for the detector along aspectral axis). For example, a shorter wavelength end of each region ofinterest can be defined by subtracting a predefined number of pixelpositions parallel to spectral axis 864 (to move to the left in thisfigure), and a longer wavelength end of each region of interest can bedefined by adding a different (or the same) number of pixel positionsparallel to spectral axis 864 (to move to the right in this figure).Similarly, the side positions of each region of interest can be set bymoving parallel to spatial axis 870 from each feature position (up ordown in this figure) by the same (or a different) number of pixelpositions. In some embodiments, spectra from a single dye can be used toset only one of the two spectral endpoints of each region of interest(e.g., the shorter wavelength endpoint using the spectra shown here).Alternatively, or in addition, one or more boundaries of each region ofinterest can be assigned based on where the signal falls off from theintensity maxima to a threshold amount (see below).

FIG. 21 shows another image representation of image data 890 collectedby an exemplary spectral imaging system. Image data 890 can describe anarray of spectral images or spectra 892 (e.g., first order images) ofwells of a sample holder holding the same mixture of two dyes in anexamination area of the imaging system. Accordingly, one of the dyes canbe used in defining the position of the shorter wavelength end of eachregion of interest, and the other dye can be used in defining theposition of the longer wavelength end of each region of interest.However, the presence of dyes producing overlapping spectral components894, 896 of each composite spectrum can make identification of maximaless reliable along a line parallel to a spectral axis 898 of the imagedata. Accordingly, a profile 900 can be generated by summing parallel tospectral axis 898 and then analyzed to identify row positions 902 withinthe image, as described above in relation to FIG. 20. A profile 904 alsocan be generated by summing orthogonal to spectral axis 898, parallel toa spatial axis 906. Profile 904 can be analyzed to identify columnpositions 908 that are intensity minima. Feature minima 910 can beidentified as the coordinates at which positions 902 and 908 intersect.Regions of interest then can be assigned based on the positions of thefeature minima, generally as described above for feature maxima.

FIG. 22 shows image data 890 of FIG. 21 being processed, in part, bythresholding to facilitate identification of features within the imagedata. Row position 902 can be identified as described above in relationto FIGS. 20 and 21. Threshold positions along each row can be identifiedby converting image data 890 to binary image data 920 according to athreshold (generally near background). Accordingly, the threshold can bepredefined or defined based on the image data. In any case, pixels withan intensity above the threshold can be set to one (or zero), indicatedat 922, and pixels with an intensity below the threshold can be set tozero (or one), indicated at 924. A resulting binary profile 926 taken atrow position 902 can be used to set positions 928 of opposing ends ofeach region of interest within the row.

In other embodiments, regions of interest can be assigned based onchanges to sample spectra produced in a kinetic assay, rather than (orin addition to) being pre-assigned via calibration standards. Forexample, image data of the samples can be collected at two or moredistinct times and changes to spectral images can be revealed bydetermining a difference image, such as by subtracting image data fromthe beginning of the reaction from image data collected at a later timepoint or endpoint of a reaction. Accordingly, regions of interest can beassigned in such subtracted image data by finding maxima, minima,transitions, thresholds, and/or the like, as described in this exampleand elsewhere in the present teachings. Further aspects of dynamicallyassigning regions of interest are described in the following patentapplication, which is incorporated herein by reference: U.S. ProvisionalPatent Application Ser. No. _(——————), filed Jun. 29, 2006, titled“TWO-DIMENSIONAL SPECTRAL IMAGING SYSTEM,” and naming Dar Bahatt andChirag Patel as inventors.

Example 8 Exemplary Use of Fiducials to Assign and/or Reposition Regionsof Interest

This example describes an exemplary use of fiducials to facilitateassigning and/or repositioning regions of interest; see FIG. 23.

FIG. 23 is a schematic view of selected portions of a spectral imagingsystem 940 that uses a fiducial 942. The fiducial can be used to correctfor variability of sample holder and/or well positioning in theexamination area of the system relative to the system optics and/or thedetector. Furthermore, in some cases, the fiducial can be used tocorrect for changes to the system optics and/or to the relation of thesystem optics to the examination area and/or detector, among others,during use, transport, storage, service, etc., of the system.

Regions of interest 944 can be assigned on a detector 946 (and thus inimage data detected by the detector) using standards 948 disposed inwells of a sample holder 950 disposed in the examination area. Fiducial942 can be disposed at the same fixed position relative to the wells ofa sample holder used to hold the standards and also relative to wellsused to hold test samples 952. Accordingly, detection of light from thefiducial on the detector, indicated at 954, can provide a referenceposition(s) 956 (standards) or 958 (samples) within the image data.

The reference position can be used to for determining where the wellsare positioned in the examination area and/or to adjust for changes inwell position produced with variability in sample holder placement intothe examination area, among others. For example, samples can be placedinto the examination area offset, indicated at 960, relative to aposition (dashed) of the sample holder during calibration and assignmentof regions of interest. Accordingly, the original detected fiducialposition, indicated as a dashed outline 964 on the sample image, isshifted translationally, indicated at 966, to new detected fiducialposition 958. Accordingly, the fiducial can be detected in an offsetposition on the detector. The regions of interest can be moved,indicated at 968, based on the offset position measured for thefiducial.

The fiducial can have any suitable structure. For example, the fiducialcan be a reflective region of the sample holder, such as a mirror toreflect excitation light, or a transmissive region of the sample holder,such as a through-hole. Alternatively, the fiducial can be luminescent,to emit light when illuminated. For example, the fiducial can be aluminescent area with a narrow band of emission, to provide awell-defined spectral image of the fiducial on the detector, generally aspectral image of the same (and/or different) diffraction order asspectral images of the wells on the detector. The fiducial can have anysuitable size, for example, a relatively small area to provide arelatively sharp fiducial image on the detector that defines a positionmore precisely.

In some examples, the fiducial image can be detected on the detector asnondispersed light from fiducial 942. Accordingly, the fiducial can beprovided by one of the wells, a row or column of the wells, and/or allof the wells.

The fiducial can have any suitable position. The fiducial be disposedwithin the array, such as between wells, outside of the array, as shownhere, or can be part of the array itself, such as when nondispersedlight serves as a positional reference or datum to facilitateidentification, particularly automatically, of higher order spectralimages. For example, the imaging system can use an algorithm to identifyregions of interest of the detector for first order spectral imagesbased on extrapolation from the position(s) of the zero ordernonspectral well images on the detector. The sample holder can have onlyone fiducial imaged by the detector, two or more fiducials (such as tocorrect for a rotational offset of the sample holder), and/or the like.

Example 9 Exemplary Methods of Sample Analysis

This example describes methods of sample analysis involving lineardecomposition of composite sample spectra; see FIGS. 24-27.

FIG. 24 shows a flowchart 1000 listing exemplary steps for performing amethod of sample analysis using a plurality of dyes. The steps listedcan be performed in any suitable order, in any suitable combination, andany suitable number of times.

Dye (or luminophore) spectra can be acquired with a two-dimensionalimaging system, indicated at 1002. The dye spectra can be acquired froma dye mixture or from dyes disposed serially in examination sites. Inparticular, the dyes can be disposed in sample holders with wells thatplace the dyes in areas large enough to create broadened dye spectrarelative to the dye spectra produced from a point or line source witheach dye.

Regions of interest for detecting the dye spectra can be assigned in adetection area of the imaging system, indicated at 1004. The regions ofinterest can be assigned based on detection of spectral images of wellsholding the dyes themselves, wells holding other standards, and/or canbe assigned based on other criteria.

A matrix describing characteristics of dye spectra within a region ofinterest can be generated, indicated at 1006, for each region ofinterest. The matrix, generally termed a calibration matrix, cancorrespond to profiles of spectral images of the dyes detected by theregion of interest. Accordingly, the distribution of signal strengthover the region of interest can be indicated for each dye spectrum.

Spectral data from samples disposed in the examination sites can bedetected (and/or obtained) based on the assigned regions of interest ofthe detection area, indicated at 1008. The spectral data for each samplecan correspond to a spectral image of the sample held by a well. Thespectral data for the sample can be obtained in exact correspondencewith a region of interest or can be shifted translationally orrotationally from the region of interest, for example, based on afiducial(s) (e.g., see Example 8).

The spectral data for each sample can be collapsed parallel to a spatialaxis of the data, indicated at 1010, generally by summing the dataparallel to the spatial axis. Collapsing the spectral data can converttwo-dimensional image data into one-dimensional data, namely, a spectralvector (a composite profile) for each sample.

The spectral vector for each sample can be decomposed, indicated at1012. In particular, the spectral vector can be a linear combination ofisolated (“pure”) dye spectra, i.e., dye spectra measured without energytransfer (or other interference) between the dyes (and/or with a non-dyequencher). The calibration matrix of step 1006 can be used for lineardecomposition, such as via multiplication by the pseudoinverse of thecalibration matrix. Relative contributions of the plurality of dyes tothe spectral vector/profile can be determined.

The relative contributions of the dyes to the spectral data can becorrelated with an aspect of the sample, indicated at 1014. The aspectcan be amount (e.g., presence/absence or level/concentration) of ananalyte(s). In some examples, the aspect can be a plurality of aspectsdetermined for the same analyte or for different analytes in the sample.Accordingly, the analysis can be a multiplexed assay of each sample,such as for the presence/absence and/or level/concentration of two ormore distinct nucleic acid analytes.

FIG. 25 shows a graph 1020 of an exemplary calibration matrix 1022 for aset of isolated dye spectra detected in the same region of interest of adetector of a spectral imaging system. A sample with each indicated dye(i.e., FAM dye, TAMRA dye, VIC dye, and ROX dye) can be disposed in thesame size/shape of well and in the same examination site of the imagingsystem for detection of the corresponding spectral image of the dyesample. The two-dimensional spectral data for each dye sample can becollapsed to one dimension, normalized (before or after collapse), andplotted according to pixel position along the spectral axis of theregion of interest.

FIG. 26 shows a graph 1040 of the calibration matrix of FIG. 25 alongwith exemplary respective collapsed spectra 1042, 1044 for a test sampledetected in the same region of interest, near the beginning (cycle 1)and end (cycle 40) of thermal cycling of the test sample. The testsample can be a reaction mixture for performing nucleic acidamplification by the polymerase chain reaction (PCR).

The sample can include a mixture of each of the dyes for which thecalibration matrix was generated. In particular, the sample can include(1) TAMRA dye as a quencher by fluorescence resonance energy transfer(FRET) of FAM dye and/or VIC dye; (2) distinct nucleic acid probes eachconjugated to FAM dye and TAMRA dye or VIC dye and TAMRA dye (or onlyone type of probe); and (3) ROX dye as an internal control (which shouldnot change substantially during the reaction).

Generation of amplification product to which each probe binds can resultin subsequent degradation of the probe by a polymerase in the reactionmixture. The polymerase can use the product at a template for furtherDNA synthesis. Degradation of the probe can reduce energy-transfer basedquenching by TAMRA. Accordingly, degradation can produce an increase inthe signal from FAM dye or VIC dye according to the probe degraded. Forexample, here, the signal for the test-sample spectrum at cycle one isstrongest at about pixel position 110 (TAMRA peak) and weaker at pixelpositions to the left of this pixel position (shorter wavelengths). Incontrast, the signal for the test-sample spectrum at cycle forty isstrongest at about pixel position 40 and relatively weaker at aboutpixel position 110, reflecting a decrease in energy transfer to TAMRA.

FIG. 27 shows an exemplary graph 1060 of the relative spectralcontributions of the four indicated dyes to the composite spectra of thePCR sample described for FIG. 26. The spectra can be detected in thesame region of interest, in real time, for example, as the sample isrepetitively cycled thermally, to promote amplification of template.Alternatively, the spectra can be detected over time as the sample isheld at the same temperature (e.g., during isothermal amplification oranother type of chemical/binding reaction). The relative contributionsof the four dyes to the composite spectra can be determined by lineardecomposition of each composite spectrum using the calibration matrix ofFIG. 25, and test-sample data at approximately the indicated cyclenumbers to generate graph 1060. Here, VIC dye and FAM dye increase inintensity due to amplification of nucleic acid analytes to which theVIC-conjugated probe and the FAM-conjugated probe can bind. In addition,the intensity of the quencher dye TAMRA drops, and ROX dye provides asteady signal.

Example 10 Exemplary Broadening of Spectra

This example describes broadening of a dye spectrum for an exemplaryspectral imaging configuration.

The system can have the following configuration. The size of a wellimage on the detector can be calculated as physical size (e.g., −0.5 to5 mm diameter), multiplied by system magnification (e.g., −0.2), to give0.1 to 1.0 mm. The size (length/width) of a pixel on the detector (e.g.,a CCD camera) can be 10 μm. Accordingly, the well image size of 0.1 to1.0 mm, which is equal to 100 to 1000 μm, can be divided by 10 μm perpixel, which gives 10 to 100 pixels. In exemplary embodiments, the wellseach have a diameter of two millimeters, the diameter (2 mm) multipliedby the optical reduction (0.20) gives an image size of 0.4 mm. Thisimage size (400 μm), with a detector of 10 μm per pixel, can give a sizeof 40 pixels over which a single wavelength of light from the well canbe distributed along a line that is parallel the spectral axis of thedetector. In exemplary embodiments, one pixel on the detectorcorresponds to about one nanometer of wavelength change in a directionparallel to the spectral axis. Accordingly, if a dye spectrum peak (froma point source) has a width of 30 nm (FWHM), it should extend over about30 pixels on the detector. However, the FWHM of 30 nm from a pointsource, should be convolved with the spectral width of the well, about40 nm, to broaden the peak from about 30 nm to 70 nm.

Example 11 Exemplary Adjustment of Optical Effects by Processing ImageData

This example describes exemplary potential advantages of processingimage data via algorithms to correct for optical distortions and/or toprovide software-level wavelength filtering.

Software algorithms for correcting image distortions can allow theoptical design of the imaging system to be relaxed (a loser tolerance)such that the system is smaller and/or cheaper. Generally, softwarecorrections are cheaper than hardware (optics) corrections.

The following relationships can be relevant in considering hardwareversus software corrections of image distortion. (A) Sphericalaberration is proportional to (1/f#)³. (B) Coma is proportional to(1/f#)²×(FOV). (C) Astigmatism and field curvature are proportional to(1/f#)×(FOV)². (D) Distortion is proportional to (FOV)³. FOV=field ofview (physical dimension); f#=f number, which equals f/D (the collectionangle of the optics is proportional to 1/f#); f=focal length; andD=effective diameter.

Distortions such as pincushion and barrel effects can come from imagingan object that is fairly large compared to the field of view of theoptical system. This means that light rays from the edges of the objectcan enter into the optical system at a relatively large angle from theoptical axis, and distortions can be a strong function of this angle.Software correction tools can allow design of a smaller and/or cheaperoptical system, where the object size is large relative to the field ofview, allowing the wells to be spaced as far as possible from oneanother. This can provide more room for dispersion and/or for morewells.

Further aspects of using algorithms to correct for optical distortionand/or to filter data according to wavelength are described in U.S.Provisional Patent Application Ser. No. 60/696,301, filed Jun. 30, 2005,which is incorporated herein by reference.

Example 12 Software-based Spectral Filters

This example describes exemplary aspects of software-based filters.

In exemplary embodiments, the pixel-to-wavelength relationship of thedetector can be about one pixel/nm and the spectral range can be˜100-150 nm. Software can be used to sum up the signal in groups ofpixels, to simulate a narrow, medium, or wide band-pass filter, that is,to approximate, in software, a “virtual” filter corresponding to ahardware filter that could be positioned in the emission filter wheel ofthe imaging system. A virtual filter can be much cheaper than a hardwarefilter.

Example 13 Selected Embodiments

This example describes selected aspects and embodiments of the presentteachings, presented as a series of numbered paragraphs.

1. A system for sample analysis, comprising: (A) a color separatorconfigured to spectrally disperse light emitted from each sample of atwo-dimensional sample array; and (B) a detector configured to detectand distinguish the dispersed light for each sample of the array.

2. The system of paragraph 1, further comprising a filter elementconfigured to reduce selectively the amount of emitted visible light ofrelatively longer wavelengths that reaches the detector.

3. The system of paragraph 2, wherein the filter element is a short-passfilter.

4. The system of paragraph 1, further comprising a sample holder to holdthe two-dimensional array of samples in a plurality of rows and columns,and wherein the rows, the columns, or both have a staggeredconfiguration.

5. The system of paragraph 1, further comprising a sample holder to holdthe two-dimensional array of samples in rows and columns arranged alongorthogonal axes in an examination, wherein the sample holder is arrangedrelative to the color separator such that the color separator dispersesemitted light spectrally to define a spectral axis of the examinationarea, and wherein the spectral axis is oblique to the each of theorthogonal axes.

6. The system of paragraph 1, further comprising a sample holder to holdthe two-dimensional array of samples, the sample holder defining a planeand including a plurality of wells configured to dispose the samples influid isolation from one another, wherein the wells are elongate in theplane to define a long axis, and wherein the wells have a greaterspacing from each other transverse to the long axis than parallel to thelong axis.

7. The system of paragraph 1, wherein the color separator disperses theemitted light for each sample into a plurality of spectral components,and wherein the detector includes an array of sensor elements, thesystem further comprising a controller in communication with thedetector and configured to relate signals from the sensor elements toparticular samples and spectral components.

8. The system of paragraph 1, further comprising a controller configuredto receive data corresponding to an image of the sample array detectedby the detector, the controller including an algorithm configured tomanipulate the data such that optical distortion of the sample array inthe image is reduced.

9. The system of paragraph 8, wherein the algorithm is configured tostretch the image diagonally, curve the image, or both.

10. The system of paragraph 8, wherein the algorithm uses a polynomialfunction to control how much the image is modified.

11. The system of paragraph 1, wherein the detector is configured suchthat nondispersed light emitted from one or more of the samples can bedetected by the detector concurrently with dispersed light from thesamples.

12. The system of paragraph 11, wherein the nondispersed light isemitted from at least one row or column of samples dispersed adjacent aperimeter of the sample array.

13. The system of paragraph 11, wherein the detector detects positionalinformation about the nondispersed light, further comprising acontroller configured to use the positional information as a referencefor relating dispersed light to particular samples and/or spectralcomponents.

14. The system of paragraph 1, further comprising a light sourceconfigured to produce light that can be directed onto a two-dimensionalarray of samples in a manner capable of producing emitted light fromeach sample of the array.

15. The system of paragraph 14, further comprising an optical relaystructure, wherein the light source includes a plurality of lightsources, and wherein the optical relay structure is configured to directlight from each light source onto a different subset of samples withinthe array.

16. The system of paragraph 15, the two-dimensional array of samplesincluding a plurality of rows, wherein the optical relay structure isconfigured to direct light from each light source onto a differentsubset of the rows.

17. The system of paragraph 15, wherein the optical relay structure isconfigured to direct light from each light source selectively onto onlyone row.

18. The system of paragraph 15, further comprising a controller incommunication with the plurality of light sources, wherein thecontroller is configured to operate the light sources electronically forserial illumination of portions of the sample array.

19. A system for sample analysis, comprising: (a) an examination areaincluding a two-dimensional array of examination sites for samples; (b)a light detector for imaging; and (c) an optical relay structure thatspectrally disperses light emitted from luminophores disposed in thetwo-dimensional array of examination sites into a correspondingtwo-dimensional array of spectra directed concurrently onto the lightdetector, the optical relay structure including one or more filtersdisposed in an optical path between the examination sites and the lightdetector and configured to substantially truncate opposing ends of eachspectrum by filtering the emitted light.

20. The system of paragraph 19, wherein the one or more filters includea short-pass and a long-pass filter.

21. The system of paragraph 19, wherein each filter substantiallyexclusively receives emitted light relative to excitation light.

22. A system for sample analysis, comprising: (a) an examination areaincluding a two-dimensional array of examination sites for samples; (b)a light detector for imaging; and (c) an optical relay structure thatspectrally disperses light emitted from luminophores disposed in thetwo-dimensional array of examination sites into a correspondingtwo-dimensional array of spectra directed concurrently onto the lightdetector such that the spectra would be substantially overlapping if notfiltered, the optical relay structure including one or more filtersdisposed in an optical path between the examination sites and the lightdetector and configured to truncate each spectrum by filtering theemitted light such that there is a substantial reduction in crosstalkbetween adjacent spectra.

23. The system of paragraph 22, wherein the optical relay structuredirects the two-dimensional array of spectra onto the light detector ina collectively nonoverlapping relation to nondispersed light from thearray of examination sites.

24. The system of paragraph 22, wherein at least one of the one or morefilters selectively restricts passage of light of relatively longerwavelengths, of relatively shorter wavelengths, or both.

25. The system of paragraph 22, wherein the one or more filters includea short-pass filter and a long-pass filter such that each spectrum istruncated at opposing ends of the spectrum.

26. The system of paragraph 25, wherein the one or more filtersselectively restrict passage of light with a wavelength of less thanabout 500 nm and greater than about 650 nm.

27. The system of paragraph 22, further comprising a sample holderincluding wells, the sample holder being configured to be received inthe examination area such that the wells are disposed in the examinationsites.

28. The system of paragraph 27, wherein the sample holder includes atleast one sealing element configured to seal each well individuallyafter the well receives a sample.

29. The system of paragraph 22, further comprising a thermal controlsystem operatively coupled to the examination area and configured tothermally regulate the examination area.

30. The system of paragraph 29, wherein the thermal control system isconfigured to thermally cycle the examination area repetitively in anautomated manner.

31. The system of paragraph 22, further comprising a light source thatilluminates each of the examination sites concurrently with excitationlight.

32. The system of paragraph 31, wherein the light source includes alight-emitting diode.

33. The system of paragraph 22, wherein the spectra of the array eachare of the same diffraction order.

34. A method of sample analysis, comprising: (a) disposing samples in atwo-dimensional array of examination sites, each sample including one ormore luminophores; (b) illuminating the samples such that the one ormore luminophores of each sample emit light; (c) dispersing the emittedlight of the samples concurrently into a corresponding two-dimensionalarray of spectra that are collectively spaced from nondispersed lightfrom the examination sites and that would be overlapping if notfiltered; (d) filtering the emitted light to substantially reducecrosstalk between adjacent spectra disposed end to end, by truncatingeach spectrum; (e) directing the array of spectra to a light detectorsuch that each spectrum of the array falls onto the light detector atthe same time; and (f) detecting an image of the array of spectra withthe light detector.

35. The method of paragraph 34, wherein the step of disposing includes astep of disposing samples including reagents for nucleic acidamplification.

36. The method of paragraph 34, wherein the step of disposing includes astep of disposing a plurality of samples each including substantiallythe same set of one or more luminophores.

37. The method of paragraph 34, wherein the step of illuminatingincludes a step of illuminating each of the samples concurrently with alight-emitting diode.

38. The method of paragraph 34, wherein the step of dispersing isperformed after the step of filtering.

39. The method of paragraph 34, wherein the step of filtering results intruncating opposing ends of each spectrum.

40. The method of paragraph 34, wherein the step of detecting includes astep of detecting a change, if any, in each spectrum over time.

41. The method of paragraph 34, wherein the step of detecting includes astep of obtaining image data corresponding to the image detected,further comprising a step of processing the image data according topre-assigned regions of a detection area to obtain spectral data foreach individual sample.

The disclosure set forth above can encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties canbe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.

1. A system for sample analysis, comprising: an examination areaincluding a two-dimensional array of examination sites; a light detectorfor imaging; an optical relay structure that spectrally disperses lightreceived from the examination sites into a corresponding two-dimensionalarray of spectra directed concurrently onto the light detector in acollectively nonoverlapping relation with nondispersed light from thetwo-dimensional array of examination sites; and a thermal control systemoperatively coupled to the examination area for regulating a temperaturethereof.
 2. The system of claim 1, further comprising a controller,wherein the thermal control system is operatively coupled to thecontroller for automated thermal control of the examination area.
 3. Thesystem of claim 1, wherein the thermal control system is configured torepetitively follow a thermal profile in an automated manner.
 4. Thesystem of claim 1, wherein the thermal control system includes a heaterand a thermally conductive member operatively coupled to the heater,further comprising a sample holder having wells and configured to bedisposed in the examination area such that the wells are disposed in theexamination sites and the sample holder is in contact with theconductive member.
 5. The system of claim 4, wherein the conductivemember and the sample holder have complementary structure that restrictslateral movement of the sample holder when the sample holder is disposedin the examination area.
 6. The system of claim 5, wherein thecomplementary structure includes one or more projections of theconductive member that are configured to be received in one or moreopenings of the sample holder.
 7. The system of claim 1, furthercomprising a sample holder having wells and configured to be disposed inthe examination area such that the wells are disposed in the examinationsites, wherein the wells have a staggered arrangement.
 8. The system ofclaim 7, wherein the wells are disposed along a set of spaced parallellines with a well-to-well spacing within each line, and wherein everyother line of wells is offset along its line by about one-half thewell-to-well spacing.
 9. The system of claim 1, further comprising asample holder having wells and configured to be disposed in theexamination area such that the wells are disposed in the examinationsites, wherein each well is at least about one millimeter in diameter.10. The system of claim 1, further comprising a sample holder havingwells and configured to be disposed in the examination area such thatthe wells are disposed in the examination sites, wherein each well has acapacity of at least about 100 nanoliters.
 11. The system of claim 1,wherein the two-dimensional array of spectra defines orthogonal axes,and wherein each spectrum extends along a spectral axis that is obliqueto each of the orthogonal axes.
 12. The system of claim 1, furthercomprising a controller operatively coupled to the light detector andconfigured to process image data collected by the light detector tocorrect for optical distortion in the image data.
 13. A method of sampleanalysis, comprising: obtaining a two-dimensional array of samples;illuminating each sample of the array such that the sample emits light;directing light from each sample to a detection area such that acorresponding two-dimensional array of spectra falls concurrently ontothe detection area; detecting the two-dimensional array of spectrarepeatedly over time to measure a change, if any, in each spectrum; anddetermining an aspect of one or more of the samples based on a measuredchange in one or more corresponding spectra.
 14. The method of claim 13,wherein the step of directing includes a step of directing light suchthat the two-dimensional array of spectra collectively is resolved fromnondispersed light from the samples.
 15. The method of claim 14, whereinthe step of directing includes a step of directing nondispersed lightfrom one or more of the samples onto the detection area.
 16. The methodof claim 13, wherein the step of obtaining a two-dimensional array ofsamples includes a step of obtaining a two-dimensional array of reactionmixtures for performing amplification of nucleic acids.
 17. A method ofsample analysis, comprising: obtaining a two-dimensional array ofsamples for performing amplification of nucleic acids; illuminating eachsample of the array concurrently such that the samples emit light;directing dispersed light from each sample to a detection area such thata corresponding two-dimensional array of spectra falls concurrently ontothe detection area; monitoring the two-dimensional array of spectra overtime to measure a change, if any, in each spectrum; and determining anaspect of one or more of the samples based on a measured change in oneor more corresponding spectra.
 18. The method of claim 17, furthercomprising a step of cycling the samples thermally through a pluralityof cycles.
 19. A system for sample analysis, comprising: a sample holderincluding a two-dimensional array of wells for receiving samples; anexamination area in which the sample holder is disposed; a lightdetector for imaging; and an optical relay structure operatively coupledto the examination area and configured to spectrally disperse lightreceived from the wells parallel to a dispersion axis into acorresponding two-dimensional array of spectra directed concurrentlyonto the light detector in a collectively nonoverlapping relation withnondispersed light from the wells, wherein each well has a sizesufficient to substantially broaden its corresponding spectrum parallelto the dispersion axis relative to a spectrum of the same spectralcomposition produced from a point source in place of the well.
 20. Thesystem of claim 19, wherein the spectrum from each well is broadened byat least about ten nanometers relative to the spectrum produced from apoint source in place of the well.
 21. The system of claim 19, whereineach well has a diameter of at least about one millimeter.
 22. Thesystem of claim 19, wherein the optical relay structure includes one ormore filters that select a range of wavelengths of the dispersed lightto direct onto the light detector.
 23. The system of claim 22, whereinthe range of wavelengths at least substantially excludes visible lightat opposing ends of a full-length visible spectrum.
 24. A system forsample analysis, comprising: a sample holder including a two-dimensionalarray of wells and one or more sealing elements configured to seal eachof the wells after the wells have received samples; an examination areain which the sample holder is positioned; a light detector for imaging;and an optical relay structure that, when the sample holder ispositioned in the examination area, spectrally disperses light receivedfrom the two-dimensional array of wells into a correspondingtwo-dimensional array of spectra directed concurrently onto the lightdetector in a collectively nonoverlapping relation with nondispersedlight from the two-dimensional array of wells.